Membrane proximal region of HIV GP41 anchored to the lipid layer of a virus-like particle vaccine

ABSTRACT

Disclosed herein are isolated immunogens including variant hepatitis B surface antigens (HBsAgs). In an example, a variant HBsAg includes a HBsAg with one or more transmembrane domains of the HBsAg replaced with a gp41 antigenic insert. The antigenic insert can include an antigenic polypeptide fragment of gp41 including the membrane proximal region of gp41 and a transmembrane membrane region of gp41. The replacement of a membrane spanning domain of HBsAg with a membrane spanning domain of gp41 anchors gp41 into HBsAg in virtually the identical orientation as on HIV virions and correctly orients the nearby MPR on the lipid layer. Thus, the disclosed variant HBsAgs display the neutralization-sensitive MPR in association with a lipid layer, while presenting it at the most immunogenic site on HBsAg. Also disclosed are uses of these variant HBsAgs, and nucleic acids encoding variant HBsAgs, such as to induce an immune response to HIV-1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/057,414, filed on Feb. 3, 2011, which is the §371 U.S. National Stageof International Application No. PCT/US2009/052724, filed on Aug. 4,2009, which was published in English under PCT Article 21(2), which inturn claims the benefit of U.S. Provisional Application No. 61/086,098,filed Aug. 4, 2008, which is incorporated herein by reference in itsentirety.

FIELD

This disclosure relates to the field of human immunodeficiency virus,specifically to the use of epitopes of glycoprotein 41 (gp41) to inducean immune response, including a protective immune response.

BACKGROUND

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. Treatments for humanimmunodeficiency virus (HIV)-infected individuals as well as thedevelopment of vaccines to protect against infection are urgentlyneeded. One difficulty has been in eliciting neutralizing antibodies tothe virus.

The HIV-1 envelope glycoproteins (gp120-gp41), which mediate receptorbinding and entry, are the major targets for neutralizing antibodies.Although the envelope glycoproteins are immunogenic and induce a varietyof antibodies, the neutralizing antibodies that are induced arestrain-specific, and the majority of the immune response is diverted tonon-neutralizing determinants (Weiss, R. A., et al., Nature, 316 (6023):69-72, 1985; Wyatt, R. and J. Sodroski, Science, 280 (5371): 1884-1888,1998). Broadly neutralizing antibodies have been isolated only rarelyfrom natural HIV infection, as only five broadly-neutralizing antibodieshave been identified to date. Three are gp41-directed (2F5, 4E10 andZ13) and the other two (b12 and 2G12) are gp120-directed. The three gp41neutralizing antibodies recognize the membrane proximal region (MPR) ofthe HIV-1 gp41 glycoprotein. The MPR region includes a series of aminoacids that lie on the HIV vsurface, just before gp41 crosses the viralmembrane. The MPR is highly hydrophobic (50% of residues arehydrophobic), and is highly conserved across many HIV clades (Zwick, M.B., et al., J Virol, 75 (22): 10892-10905, 2001). Recently thehydrophobic context of MPR and the presence of lipid membrane were shownto be important for the optimal binding of 2F5 and 4E10 antibodies(Ofek, G., et al., J Virol, 78 (19): 10724-37, 2004).

To date, immunization with conserved membrane proximal elements or thecore 2F5 epitope in a number of contexts has failed to elicit broadlyneutralizing antibodies (Coeffier, E., et al., Vaccine, 19 (7-8):684-693, 2000; Eckhart, L., et al., J Gen Virol, 77 (Pt 9): 2001-2008,1996; Ernst, W., et al., Nucleic Acids Res, 26 (7): 1718-1723, 1998; Ho,J., et al., Vaccine, 20 (7-8): 1169-1180, 2002; Liang, X., et al.,Epitop Vaccine, 17 (22): 2862-2872, 1999; Liao, M., et al., Peptides, 21(4): 463-468, 2000; Xiao, Y., et al., Immunol Invest, 29 (1): 41-50,2000). Thus, there remains a need to identify HIV antigens that can beused to induce a protective immune response.

SUMMARY

Historically, compositions used to produce an immune response againstviral antigens included live-attenuated or chemically inactivated formsof the virus. However, this approach has limited utility when used forhuman immunodeficiency virus. Disclosed herein is the use of theimmunogenic hepatitis B surface antigen (HBsAg) platform to arrayepitopes from the conserved, neutralization-sensitive membrane proximalregion (MPR) of HIV-1, and the use of this platform to induce an immuneresponse to HIV-1.

In one embodiment, isolated immunogens including variant HBsAgs aredisclosed. In an example, a variant HBsAg includes an HBsAg with one ormore transmembrane domains of the HBsAg replaced with a gp41transmembrane domain and/or one or more gp41 MPRs. For example, avariant HBsAg can include a gp41 antigenic insert that includes anantigenic polypeptide fragment of gp41 including the MPR of gp41 and atransmembrane domain of gp41. The replacement of a membrane spanningdomain of HBsAg with a membrane spanning domain of gp41 anchors gp41into HBsAg in virtually the identical orientation as on HIV virions andcorrectly orients the nearby MPR on the lipid layer. Thus, the disclosedvariant HBsAgs display the neutralization-sensitive MPR in associationwith a lipid layer, while presenting it at the most immunogenic site onHBsAg. Also disclosed are uses of these variant HBsAgs, and nucleicacids encoding variant HBsAgs, such as to induce an immune response toHIV-1.

In some embodiments, an isolated immunogen includes a variant HBsAg withone or more transmembrane domains of the HBsAg replaced with a gp41antigenic insert. The gp41 antigenic insert includes (a) an antigenicpolypeptide fragment of gp41, such as an antigenic polypeptide gp41fragment with the amino acid sequence of SEQ ID NO: 1, and (b) atransmembrane domain of gp41, such as a transmembrane spanning gp41region with the amino acid sequence set forth in SEQ ID NO: 25 (in whichX₁, X₂, X₃, and X₄ are any amino acid and X₅, X₆, and X₇ are anyhydrophobic amino acid). In one example, the antigenic polypeptidefragment of gp41 is between 28 and 150 amino acids in length and themembrane spanning region of gp41 is between 22 and 40 amino acids inlength.

In one particular embodiment, an isolated immunogen includes a variantHBsAg in which the first transmembrane spanning domain of the HBsAg isreplaced by a gp41 antigenic insert. For example, the gp41 antigenicinsert replaces amino acid residues 1-35 of SEQ ID NO: 31. In anotherexample, the gp41 antigenic insert replaces amino acid residues 1-32 ofSEQ ID NO: 31. In yet another example, the gp41 antigenic insertreplaces amino acid residues 1-29 of SEQ ID NO: 31. In a particularexample, an isolated immunogen including a variant HBsAg in which thefirst transmembrane spanning domain of the HBsAg is replaced by a gp41antigenic insert has the amino acid sequence set forth as SEQ ID NO: 29.

In another particular embodiment, an isolated immunogen includes avariant HBsAg in which the third transmembrane spanning domain of theHBsAg is replaced by a gp41 antigenic insert. For example, the gp41antigenic insert replaces amino acid residues 150-190 of SEQ ID NO: 31.In another example, the gp41 antigenic insert replaces amino acidresidues 153-187 of SEQ ID NO: 31. In yet another example, the gp41antigenic insert replaces amino acid residues 156-185 of SEQ ID NO: 31.In a particular example, an isolated immunogen including a variant HBsAgin which the third transmembrane spanning domain of the HBsAg isreplaced by a gp41 antigenic insert has the amino acid sequence setforth as SEQ ID NO: 57.

In an even more particular embodiment, an isolated immunogen includes avariant HBsAg in which the first and the third transmembrane spanningdomains of the HBsAg are replaced by a gp41 antigenic insert. Forexample, the gp41 antigenic insert replaces amino acid residues 1-35 and150-190 of SEQ ID NO: 31. In another example, the gp41 antigenic insertreplaces amino acid residues 1-32 and 153-187 of SEQ ID NO: 31. In yetanother example, the gp41 antigenic insert replaces amino acid residues1-29 and 156-185 of SEQ ID NO: 31. In a particular example, an isolatedimmunogen includes a variant HBsAg in which the third transmembranespanning domain of the HBsAg is replaced by a gp41 antigenic insert thathas the amino acid sequence set forth as SEQ ID NO: 58.

Isolated nucleic acid molecules encoding the variant HBsAgs are alsoprovided, as well as host cells transformed with the nucleic acidmolecules and viral-like particles produced by the transformed hostcells. The variant HBsAgs may further include elements, such as one ormore HIV-specific T-helper cell epitopes. Viral-like particles includingthe variant HBsAgs are also provided herein. Compositions comprising theviral-like particles are also provided.

The disclosed isolated immunogen including a variant HBsAg can be usedto induce an immune response, such as a protective immune response, whenintroduced into a subject. The isolated immunogen can also be used inassays to diagnose an HIV infection. Thus, methods are provided forinhibiting HIV infection in a subject, for inducing an immune responseto HIV in a subject, for diagnosing HIV infection in a subject, and foridentifying a B cell that produces antibodies that bind to gp41.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF SEQUENCES

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. All sequence database accession numbers referencedherein are understood to refer to the version of the sequence identifiedby that accession number as it was available on the designated date. Inthe accompanying sequence listing:

SEQ ID NO: 1 is a consensus amino acid sequence for the membraneproximal region (MPR) of gp41 of HIV-1. An X represents specific aminoacids where alterations can be tolerated.

SEQ ID NO: 2 is a consensus amino acid sequence based on each cladeconsensus sequence of the MPR region from HIV-1.

SEQ ID NO: 3 is the ancestral amino acid sequence of the MPR region fromHIV-1 clade M. This sequence is also the consensus amino acid sequenceof the MPR region from HIV-1 clade AG.

SEQ ID NO: 4 is the consensus amino acid sequence of the MPR region fromHIV-1 clade A1. This sequence is also the ancestral amino acid sequenceof the MPR region from HIV-1 clade A1.

SEQ ID NO: 5 is the consensus amino acid sequence of the MPR region fromHIV-1 clade A2.

SEQ ID NO: 6 is the consensus amino acid sequence of the MPR region fromHIV-1 clade B. This sequence is also the ancestral amino acid sequenceof the MPR region from HIV-1 clade B.

SEQ ID NO: 7 is the consensus amino acid sequence of the MPR region fromHIV-1 clade C.

SEQ ID NO: 8 is the ancestral amino acid sequence of the MPR region fromHIV-1 clade C.

SEQ ID NO: 9 is the consensus amino acid sequence of the MPR region fromHIV-1 clade D.

SEQ ID NO: 10 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade F1.

SEQ ID NO: 11 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade F2.

SEQ ID NO: 12 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade G.

SEQ ID NO: 13 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade H.

SEQ ID NO: 14 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade AE.

SEQ ID NO: 15 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade AB.

SEQ ID NO: 16 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 04CPX.

SEQ ID NO: 17 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 06CPX.

SEQ ID NO: 18 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 08BC.

SEQ ID NO: 19 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 10CD.

SEQ ID NO: 20 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 11CPX.

SEQ ID NO: 21 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 12BF.

SEQ ID NO: 22 is the consensus amino acid sequence of the MPR regionfrom HIV-1 clade 14BG.

SEQ ID NO: 23 is an exemplary amino acid sequence of an MPR regioninserted into a disclosed HBsAg construct (TM12).

SEQ ID NO: 24 an exemplary amino acid sequence of an MPR region insertedinto a disclosed HBsAg construct (TM14, TM16 or TM20)

SEQ ID NO: 25 is a consensus amino acid sequence for the transmembraneregion of gp41. An X represents any hydrophobic amino acid.

SEQ ID NOs: 26-28 are amino acid sequences for a transmembrane domain ofgp41.

SEQ ID NO: 29 is an amino acid sequence for a disclosed isolatedimmunogen in which the first transmembrane domain of hepatitis B surfaceantigen is replaced with the MPR and transmembrane domain of gp41.

SEQ ID NO: 30 is a leader sequence from influenza hemagglutininMKTIIALSYIFCLVFAQDLPGNDNNS.

SEQ ID NO: 31 is an amino acid sequence of an exemplary wildtype HBsAg.

SEQ ID NO: 32 is an example of a nucleotide sequence for a T helper cellepitope.

SEQ ID NO: 33 is an example of an amino acid sequence for a T helpercell epitope.

SEQ ID NO: 34 is the CAAX amino acid sequence, where C is cystein, A isan aliphatic amino acid and X is any amino acid.

SEQ ID NO: 35 is the core amino acid sequence of the 2F5 epitope.

SEQ ID NO: 36 is the core amino acid sequence of the 4E10 epitope.

SEQ ID NO: 37 is the linker sequence GPGP.

SEQ ID NO: 38 is a forward primer for amplification of the HBsAg.

SEQ ID NO: 39 is a reverse primer for amplification of the HBsAg.

SEQ ID NO: 40 is a forward primer for amplification of MPR.

SEQ ID NO: 41 is a reverse primer for amplification of MPR.

SEQ ID NO: 42 is a reverse primer for amplification of MPR-Foldon.

SEQ ID NO: 43 is a forward primer for amplification of C-heptad.

SEQ ID NO: 44 is a reverse primer for amplification of MPR-Tm5.

SEQ ID NO: 45 is a reverse primer for amplification of MPR-Tm10.

SEQ ID NO: 46 is a reverse primer for amplification of MPR-Tm15.

SEQ ID NO: 47 is a reverse primer for amplification of MPR-Tm23.

SEQ ID NO: 48 is a forward primer for amplification of the MPR regionwith AgeI.

SEQ ID NO: 49 is a reverse primer for amplification of the MPR regionwith AgeI.

SEQ ID NO: 50 is a forward primer for amplification of the MPR regionwith AgeI.

SEQ ID NO: 51 is a reverse primer for amplification of the MPR regionwith AgeI.

SEQ ID NO: 52 is a forward primer for amplification of the MPR regionwith HBsAg (MPRSAG or MPR-N-term).

SEQ ID NO: 53 is a reverse primer for amplification of the MPR regionwith HBsAg (MPRSAG or MPR-N-term).

SEQ ID NO: 54 is a forward primer for amplification of SAGMPR-R1 (HBsAgat the N-terminus of MPR).

SEQ ID NO: 55 is a reverse primer for amplification of SAGMPR-R1 (HBsAgat the N-terminus of MPR).

SEQ ID NO: 56 is an amino acid sequence of a disclosed variant HbsAgconstruct (34) in which an MPR is inserted between a second and thirddomain in the variant HBsAg.

SEQ ID NO: 57 is an amino acid sequence for a disclosed isolatedimmunogen in which the third transmembrane domain of hepatitis B surfaceantigen is replaced with the MPR and transmembrane domain of gp41.

SEQ ID NO: 58 is an amino acid sequence for a disclosed isolatedimmunogen in which the first and third transmembrane domains ofhepatitis B surface antigen are each replaced with the MPR andtransmembrane domain of gp41.

SEQ ID NO: 59 is a nucleic acid sequence for a disclosed isolatedimmunogen in which the third transmembrane domain of HBsAg is replacedwith the MPR and transmembrane domain of gp41.

SEQ ID NO: 60 is an amino acid sequence of the MPR region in the TM32 orTM32F constructs.

SEQ ID NO: 61 is an amino acid sequence of the MPR region in the TM34construct.

SEQ ID NO: 62 is an amino acid sequence of a disclosed variant HbsAgconstruct (TM16+34) in which the first domain is replaced with a MPR andtransmembrane domain of gp41 and an additional MPR is inserted between asecond and third domain in the variant HBsAg.

SEQ ID NO: 63 is an amino acid sequence of a disclosed variant HbsAgconstruct (32F) in which four MPRs are inserted between a second andthird domain in the variant HBsAg.

SEQ ID NO: 64 is an amino acid sequence of a disclosed variant HbsAgconstruct (TM16+32F) in which the first domain is replaced with a MPRand transmembrane domain of gp41 and four additional MPRs are insertedbetween a second and third domain in the variant HBsAg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of virus-like particles expressingthe MPR of gp41 demonstrating the MPR anchored to a lipid membrane.

FIG. 2A is a schematic illustrating the location of MPR in gp41 and avariant HBsAg in which the first domain of HBsAg was replaced with themembrane spanning domain of gp41 that allows the MPR to be correctlyoriented in relation to the lipid and anchors the MPR from gp41 withinHBsAg.

FIG. 2B is a schematic illustrating HBsAg including four transmembranedomains.

FIG. 3A is a diagram illustrating the insertion of various gp41 membranespanning domains into the first membrane spanning domain of HBsAg.

FIG. 3B is a digital image of a gel illustrating MPR expression in HBsAgwhen gp41 is substituted for the beginning of the first transmembranedomain of HBsAg (TM12) or substituted for the entire first transmembranedomain of HBsAg (TM16). Substitution for approximately half of the firsttransmembrane domain of HBsAg (TM14) did not result in detectable MPRexpression.

FIG. 4 is a digital image of a gel showing purification of MPR particlesby hydrophobic interaction chromatography.

FIG. 5 is a pair of digital images of gels illustrating the purity ofHBsAg when a gp41 membrane spanning domain is substituted for the firsttransmembrane domain of HBsAg (TM16).

FIG. 6 is a pair of digital images of particles detected by electronmicroscopy illustrating the size of variant HBsAgs in which the firsttransmembrane domain is substituted with a gp41 membrane spanning domainand that such variants are capable of particle formation.

FIG. 7 is a pair of graphs illustrating successful HBsAg particleassembly for variant HBsAgs in which the third transmembrane domain issubstituted with a gp41 membrane spanning domain as demonstrated by sizeexclusion chromatography and cesium chloride gradient.

FIG. 8. is digital image of gel illustrating purification of a variantHBsAg (MPRS) by a methyl HIC column in which the variant HBsAg includesa gp41 insert following the fourth domain of the HBsAg (MPRS).

FIG. 9A is a series of schematics illustrating exemplary variant HBsAgconstructs that assemble particles based on sedimentation studies (TM12,DA31-34, MPR, TM16, TM20 and MPRS) and electron microscopy (TM16).

FIG. 9B is a pair of schematics illustrating additional exemplaryvariant HBsAg constructs that assemble particles.

FIG. 10 is an illustration of an exemplary gp41 antigenic insertillustrating the MPR (SEQ ID NO: 1) and membrane spanning domain (SEQ IDNO: 25) of various inserts.

FIG. 11 is a pair of graphs illustrating the size and expression of ofHBsAg constructs with either single or double inserts as tested bysedimentation in sucrose gradients.

FIG. 12 is a series of digital images illustrating successful particleformation of variant HBsAgs in which two domains with the HBsAg aresubstituted (each specific construct is illustrated by a schematic beloweach representative image).

FIG. 13 is a graph illustrating antigenicity of various disclosedvariant HBsAg constructs is dependent upon the valency of the constructswherein divalent insertion of MPR determinants into TM16+31/34 had asynergistic effect on antibody binding.

FIG. 14 is a pair of bar graphs illustrating the immunogenicity of thedisclosed multivalent MPR particles.

DETAILED DESCRIPTION I. Abbreviations and Terms

AIDS: acquired immune deficiency syndrome

Gp41: glycoprotein 41

HBsAg: hepatitis B surface antigen

HIV: human immunodeficiency virus

MPR: membrane proximal region

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting

To facilitate review of the various embodiments of this disclosure, thefollowing explanations of specific terms are provided:

Adjuvant: A vehicle used to enhance antigenicity; such as a suspensionof minerals (alum, aluminum hydroxide, or phosphate) on which antigen isadsorbed; or water-in-oil emulsion in which antigen solution isemulsified in mineral oil (Freund incomplete adjuvant), sometimes withthe inclusion of killed mycobacteria (Freund's complete adjuvant) tofurther enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunstimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. No. 6,194,388; U.S. Pat. No. 6,207,646; U.S. Pat.No. 6,214,806; U.S. Pat. No. 6,218,371; U.S. Pat. No. 6,239,116; U.S.Pat. No. 6,339,068; U.S. Pat. No. 6,406,705; and U.S. Pat. No.6,429,199).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term is usedinterchangeably with the term “immunogen.” The term “antigen” includesall related antigenic epitopes. An “antigenic polypeptide” is apolypeptide to which an immune response, such as a T cell response or anantibody response, can be stimulated. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids (linear) or noncontiguousamino acids juxtaposed by tertiary folding of an antigenic polypeptide(conformational). Epitopes formed from contiguous amino acids aretypically retained on exposure to denaturing solvents whereas epitopesformed by tertiary folding are typically lost on treatment withdenaturing solvents. Normally, a B-cell epitope will include at leastabout 5 amino acids but can be as small as 3-4 amino acids. A T-cellepitope, such as a CTL epitope, will include at least about 7-9 aminoacids, and a helper T-cell epitope at least about 12-20 amino acids.Normally, an epitope will include between about 5 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The amino acids are in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography andmulti-dimensional nuclear magnetic resonance spectroscopy. The term“antigen” denotes both subunit antigens, (for example, antigens whichare separate and discrete from a whole organism with which the antigenis associated in nature), as well as killed, attenuated or inactivatedbacteria, viruses, fungi, parasites or other microbes. Antibodies suchas anti-idiotype antibodies, or fragments thereof, and synthetic peptidemimotopes, which can mimic an antigen or antigenic determinant, are alsocaptured under the definition of antigen as used herein. Similarly, anoligonucleotide or polynucleotide which expresses an antigen orantigenic determinant in vivo, such as in gene therapy and DNAimmunization applications, is also included in the definition of antigenherein.

An “antigen,” when referring to a protein, includes a protein withmodifications, such as deletions, additions and substitutions (generallyconservative in nature) to the native sequence, so long as the proteinmaintains the ability to elicit an immunological response, as definedherein. These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the antigens.

Antigen Delivery Platform or Epitope Mounting Platform: In the contextof the present disclosure, the terms “antigen delivery platform” and“epitope mounting platform” refer to a macromolecular complex includingone or more antigenic epitopes. Delivery of an antigen (including one ormore epitopes) in the context of an epitope mounting platform enhances,increases, ameliorates or otherwise improves a desired antigen-specificimmune response to the antigenic epitope(s). The molecular constituentsof the antigen delivery platform may be antigenically neutral or may beimmunologically active, that is, capable of generating a specific immuneresponse. Nonetheless, the term antigen delivery platform is utilized toindicate that a desired immune response is generated against a selectedantigen that is a component of the macromolecular complex other than theplatform polypeptide to which the antigen is attached. Accordingly, theepitope mounting platform is useful for delivering a wide variety ofantigenic epitopes, including antigenic epitopes of pathogenic organismssuch as bacteria and viruses. The antigen delivery platform of thepresent disclosure is particularly useful for the delivery of complexpeptide or polypeptide antigens, which may include one or many distinctepitopes.

Amplification: A technique that increase the number of molecules in aspecimen. Amplification of a nucleic acid molecule (e.g., a DNA or RNAmolecule) refers to use of a technique that increases the number ofcopies of a nucleic acid molecule in a specimen. An example ofamplification is the polymerase chain reaction (PCR), in which abiological sample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, that is, molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(H1) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(H1) domains; (iii) an Fv fragment consisting of the V_(L) andV_(H) domains of a single arm of an antibody, (iv) a dAb fragment (Wardet al., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v)an isolated complimentarity determining region (CDR); and (vi) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Methods of producing polyclonal and monoclonal antibodies are known tothose of ordinary skill in the art, and many antibodies are available.See, e.g., Coligan, Current Protocols in Immunology Wiley/Greene, N.Y.,1991; and Harlow and Lane, Antibodies: A Laboratory Manual Cold SpringHarbor Press, N.Y., 1989; Stites et al., (eds.) Basic and ClinicalImmunology (4th ed.) Lange Medical Publications, Los Altos, Calif., andreferences cited therein; Goding, Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y. 1986; and Kohler andMilstein, Nature 256: 495-497, 1975. Other suitable techniques forantibody preparation include selection of libraries of recombinantantibodies in phage or similar vectors. See, Huse et al., Science 246:1275-1281, 1989; and Ward et al., Nature 341: 544-546, 1989. “Specific”monoclonal and polyclonal antibodies and antisera (or antiserum) willusually bind with a K_(D) of at least about 0.1 μM, preferably at leastabout 0.01 μM or better, and most typically and preferably, 0.001 μM orbetter.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. No.4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694;EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J.Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed), Antibody Engineering, 2^(nd) Edition Freeman andCompany, N.Y., 1995; McCafferty et al., Antibody Engineering, APractical Approach, IRL at Oxford Press, Oxford, England, 1996, and PaulAntibody Engineering Protocols Humana Press, Towata, N.J., 1995.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antigenic polypeptide fragment: A polypeptide that is antigenic. In anexample, an antigenic polypeptide fragment includes a MPR of gp41, suchas a MPR with an amino acid sequence set forth in SEQ ID NO: 1.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease a desiredactivity of a protein or polypeptide. For example, in the context of thepresent disclosure, a conservative amino acid substitution does notsubstantially alter or decrease the immunogenicity of an antigenicepitope. Similarly, a conservative amino acid substitution does notsubstantially affect the structure or, for example, the stability of aprotein or polypeptide. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity or antigenicity or substantially alter astructure, such as a secondary or tertiary structure, of a protein orpolypeptide.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is typically synthesized in the laboratory byreverse transcription from messenger RNA extracted from cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to a condition induced by a viral orother pathogen. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of true positives).The “specificity” of a diagnostic assay is 1 minus the false positiverate, where the false positive rate is defined as the proportion ofthose without the disease who test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis. “Prognostic” is the probability of development (or forexample, the probability of severity) of a pathologic condition, such asa symptom induced by a viral infection or other pathogenic organism, orresulting indirectly from such an infection.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, that is, thatelicit a specific immune response. An antibody specifically binds aparticular antigenic epitope on a polypeptide. Epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and multi-dimensionalnuclear magnetic resonance spectroscopy. See, e.g., “Epitope MappingProtocols” in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996). In one embodiment, an epitope binds an MHC molecule, e.g., anHLA molecule or a DR molecule. These molecules bind polypeptides havingthe correct anchor amino acids separated by about eight or nine aminoacids

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus, expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (typically, ATG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

Glycoprotein 41 (gp41): An HIV-1 envelope glycoprotein that mediatesreceptor binding and HIV entry into a cell. Gp41 includes a MPR and atransmembrane spanning domain. Gp41 is immunogenic and induces a varietyof neutralizing antibodies, such as neutralizing antibodies directed to2F5, 4E10 and Z13. These three gp41 neutralizing antibodies recognizethe MPR of the HIV-1 gp41 glycoprotein.

Gp41 antigenic insert: A peptide fragment that includes a membraneproximal region of gp41 and a transmembrane domain of gp41. In anexample, the membrane proximal region (also referred to as the antigenicpolypeptide fragment) of gp41 includes the amino acid sequence of SEQ IDNO: 1 and a transmembrane domain of gp41 including the amino acidsequence set forth as SEQ ID NO: 25. For example, the antigenicpolypeptide fragment of gp41 is between 28 and 150 amino acids in lengthand the transmembrane domain of gp41 is between 22 and 40 amino acids inlength and wherein the transmembrane domain of gp41 is C-terminal to theantigenic polypeptide fragment of gp41.

Hepatitis B Surface Antigen (HBsAg): HBsAg is composed of 3polypeptides, preS1, preS2 and S that are produced from alternativetranslation start sites. The surface proteins have many functions,including attachment and penetration of the virus into hepatocytes atthe beginning of the infection process. The surface antigen is aprincipal component of the hepatitis B envelope. HBsAg has four membranespanning domains. As used herein, a variant HBsAg is a HBsAg thatincludes a MPR from gp41. In a particular example, a variant HBsAgincludes a MPR and a membrane spanning domain from gp41. In certainexamples, a variant HBsAg has an amino acid sequence set forth by SEQ IDNOs: 29, 56-58, 62-64 or an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequencesimilarity, such as 80%, 82%, 85%, 87%, 90%, 93%, 95% or 98% sequencesimilarity with such sequences.

Host cells: Cells in which a polynucleotide, for example, apolynucleotide vector or a viral vector, can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Human Immunodeficiency Virus (HIV): A virus, known to cause AIDS, thatincludes HIV-1 and HIV-2. HIV-1 is composed of two copies ofsingle-stranded RNA enclosed by a conical capsid including the viralprotein p24, typical of lentiviruses. The capsid is surrounded by aplasma membrane of host-cell origin.

The envelope protein of HIV-1 is made up of a glycoprotein called gp160.The mature, virion associated envelope protein is a trimeric moleculecomposed of three gp120 and three gp41 subunits held together by weaknoncovalent interactions. This structure is highly flexible andundergoes substantial conformational changes upon gp120 binding with CD4and chemokine coreceptors, which leads to exposure of the fusionpeptides of gp41 that insert into the target cell membrane and mediateviral entry. Following oligomerization in the endoplasmic reticulum, thegp160 precursor protein is cleaved by cellular proteases and istransported to the cell surface. During the course of HIV-1 infection,the gp120 and gp41 subunits are shed from virions and virus-infectedcells due to the noncovalent interactions between gp120 and gp41 andbetween gp41 subunits.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In some cases, the response isspecific for a particular antigen (that is, an “antigen-specificresponse”). In some cases, an immune response is a T cell response, suchas a CD4+ response or a CD8+ response. Alternatively, the response is aB cell response, and results in the production of specific antibodies.For purposes of the present invention, a “humoral immune response”refers to an immune response mediated by antibody molecules, while a“cellular immune response” is one mediated by T-lymphocytes and/or otherwhite blood cells. A “protective immune response” is an immune responsethat inhibits a detrimental function or activity (such as a detrimentaleffect of a pathogenic organism such as a virus), reduces infection by apathogenic organism (such as, a virus), or decreases symptoms thatresult from infection by the pathogenic organism. A protective immuneresponse can be measured, for example, by the inhibition of viralreplication or plaque formation in a plaque reduction assay orELISA-neutralization assay (NELISA), or by measuring resistance to viralchallenge in vivo.

An immunogenic composition can induce a B cell response. The ability ofa particular antigen to stimulate a B cell response can be measured bydetermining if antibodies are present that bind the antigen. In oneexample, neurtralizing antibodies are produced.

One aspect of cellular immunity involves an antigen-specific response bycytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigensthat are presented in association with proteins encoded by the majorhistocompatibility complex (MHC) and expressed on the surface of cells.CTLs help induce and promote the destruction of intracellular microbes,or the lysis of cells infected with such microbes. Another aspect ofcellular immunity involves an antigen-specific response by helperT-cells. Helper T-cells act to help stimulate the function, and focusthe activity of, nonspecific effector cells against cells displayingpeptide antigens in association with MHC molecules on their surface. A“cellular immune response” also refers to the production of cytokines,chemokines and other such molecules produced by activated T-cells and/orother white blood cells, including those derived from CD4+ and CD8+T-cells.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See, forexample, Erickson et al. (1993) J. Immunol. 151:4189-4199; Doe et al.(1994) Eur. J. Immunol. 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (for example, by the tetramer technique)(reviewed by McMichael and O'Callaghan (1998) J. Exp. Med.187(9)1367-1371; Mcheyzer-Williams et al. (1996) Immunol. Rev. 150:5-21;Lalvani et al. (1997) J. Exp. Med. 186:859-865).

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or gamma-delta T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived.

Immunogenic composition: A composition comprising at least one epitopeof a virus, or other pathogenic organism, that induces a measurable CTLresponse, or induces a measurable B cell response (for example,production of antibodies that specifically bind the epitope). It furtherrefers to isolated nucleic acids encoding an immunogenic epitope ofvirus or other pathogen that can be used to express the epitope (andthus be used to elicit an immune response against this polypeptide or arelated polypeptide expressed by the pathogen). For in vitro use, theimmunogenic composition may consist of the isolated nucleic acid,protein or peptide. For in vivo use, the immunogenic composition willtypically include the nucleic acid, protein or peptide inpharmaceutically acceptable carriers or excipients, and/or other agents,for example, adjuvants. An immunogenic polypeptide (such as an antigenicpolypeptide), or nucleic acid encoding the polypeptide, can be readilytested for its ability to induce a CTL or antibody response byart-recognized assays.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, for example, other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, affinity tags, enzymatic linkages, and radioactive isotopes. Anaffinity tag is a peptide or polypeptide sequence capable ofspecifically binding to a specified substrate, for example, an organic,non-organic or enzymatic substrate or cofactor. A polypeptide includinga peptide or polypeptide affinity tag can typically be recovered, forexample, purified or isolated, by means of the specific interactionbetween the affinity tag and its substrate. An exemplary affinity tag isa poly-histidine (e.g., six-histidine) affinity tag which canspecifically bind to non-organic metals such as nickel and/or cobalt.Additional affinity tags are well known in the art.

Linking peptide: A linking peptide (or linker sequence) is an amino acidsequence that covalently links two polypeptide domains. Linking peptidescan be included between the rotavirus NSP2 polypeptide and an antigenicepitope to provide rotational freedom to the linked polypeptide domainsand thereby to promote proper domain folding. Linking peptides, whichare generally between 2 and 25 amino acids in length, are well known inthe art and include, but are not limited to the amino acid sequencesglycine-proline-glycine-proline (GPGP)(SEQ ID NO: 37) andglycine-glycine-serine (GGS), as well as the glycine(4)-serine spacerdescribed by Chaudhary et al., Nature 339:394-397, 1989. In some casesmultiple repeats of a linking peptide are present.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells. “T lymphocytes” or “T cells” are non-antibody producinglymphocytes that constitute a part of the cell-mediated arm of theimmune system. T cells arise from immature lymphocytes that migrate fromthe bone marrow to the thymus, where they undergo a maturation processunder the direction of thymic hormones. Here, the mature lymphocytesrapidly divide increasing to very large numbers. The maturing T cellsbecome immunocompetent based on their ability to recognize and bind aspecific antigen. Activation of immunocompetent T cells is triggeredwhen an antigen binds to the lymphocyte's surface receptors. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

Mammal: This term includes both human and non-human mammals unlessotherwise specified. Similarly, the term “subject” includes both humanand veterinary subjects.

Membrane proximal region (MPR) or membrane proximal external region(MPER) of gp41: A region that is immediately N-terminal of thetransmembrane region of gp41. The MPR is highly hydrophobic (50% ofresidues are hydrophobic) and is highly conserved across many HIV clades(Zwick, M. B., et al., J Virol, 75 (22): p. 10892-905, 2001). Theconserved MPR of HIV-1 gp41 is a target of two broadly neutralizinghuman monoclonal antibodies, 2F5 and 4E10. The core of the 2F5 epitopehas been shown to be ELDKWAS (SEQ ID NO: 35). With this epitope, theresidues D, K, and W were found to be most critical for recognition by2F5. The core of the 4E10 epitope, NWFDIT (SEQ ID NO: 36), maps justC-terminal to the 2F5 epitope on the gp41 ectodomain.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (“ORF”): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a polypeptide (peptide orprotein).

Operatively linked: A first nucleic acid sequence is operatively linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operatively linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operatively linked DNA sequences arecontiguous and, where necessary to join two protein-coding regions, inthe same reading frame, for example, two polypeptide domains orcomponents of a fusion protein.

Pharmaceutically acceptable carriers and/or pharmaceutically acceptableexcipients: The pharmaceutically acceptable carriers or excipients ofuse are conventional. Remington's Pharmaceutical Sciences, by E. W.Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describescompositions and formulations suitable for pharmaceutical delivery ofthe polypeptides and polynucleotides disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

A “therapeutically effective amount” is a quantity of a composition usedto achieve a desired effect in a subject. For instance, this can be theamount of the composition necessary to inhibit viral (or other pathogen)replication or to prevent or measurably alter outward symptoms of viral(or other pathogenic) infection. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations (for example, in lymphocytes) that has been shown toachieve an in vitro effect.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (for example, glycosylation orphosphorylation), such as a protein or a fragment or subsequence of aprotein. The term “peptide” is typically used to refer to a chain ofamino acids of between 3 and 30 amino acids in length. For example animmunologically relevant peptide may be between about 7 and about 25amino acids in length, e.g., between about 8 and about 10 amino acids.

In the context of the present disclosure, a polypeptide can be a fusionprotein comprising a plurality of constituent polypeptide (or peptide)elements. Typically, the constituents of the fusion protein aregenetically distinct, that is, they originate from distinct geneticelements, such as genetic elements of different organisms or fromdifferent genetic elements (genomic components) or from differentlocations on a single genetic element, or in a different relationshipthan found in their natural environment. Nonetheless, in the context ofa fusion protein the distinct elements are translated as a singlepolypeptide. The term monomeric fusion protein (or monomeric fusionprotein subunit) is used synonymously with such a single fusion proteinpolypeptide to clarify reference to a single constituent subunit wherethe translated fusion proteins assume a multimeric tertiary structure.

Specifically, in an embodiment, a monomeric fusion protein subunitincludes in an N-terminal to C-terminal direction: a viral NSP2polypeptide; a linear linking peptide; and an antigenic polypeptide orepitope translated into a single polypeptide monomer. A plurality (forexample, 4, 8, 12 or 16) of monomeric fusion protein subunitsself-assembles into a multimeric ring structure.

Preventing or treating an infection: Inhibiting infection by a pathogensuch as a virus, such as a lentivirus, or other virus, refers toinhibiting the full development of a disease either by avoiding initialinfection or inhibiting development of the disease process once it isinitiated. For example, inhibiting a viral infection refers to lesseningsymptoms resulting from infection by the virus, such as preventing thedevelopment of symptoms in a person who is known to have been exposed tothe virus, or to lessening virus number or infectivity of a virus in asubject exposed to the virus. “Treatment” refers to a therapeutic orprophylactic intervention that ameliorates or prevents a sign or symptomof a disease or pathological condition related to infection of a subjectwith a virus or other pathogen.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides, for example, a nucleotidesequence of about 15 nucleotides or more in length. Primers may beannealed to a complementary target DNA strand by nucleic acidhybridization to form a hybrid between the primer and the target DNAstrand, and then extended along the target DNA strand by a DNApolymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, for example, by the polymerase chain reaction(PCR) or other nucleic-acid amplification methods known in the art. Oneof skill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 20 consecutive nucleotides will anneal to a target with ahigher specificity than a corresponding primer of only about 15nucleotides. Thus, in order to obtain greater specificity, probes andprimers may be selected that comprise 20, 25, 30, 35, 40, 50 or moreconsecutive nucleotides.

Promoter: A promoter is a minimal sequence sufficient to directtranscription. Also included are those promoter elements which aresufficient to render promoter-dependent gene expression controllable forcell-type specific, tissue-specific, or inducible by external signals oragents; such elements may be located in the 5′ or 3′ regions of thegene. Both constitutive and inducible promoters are included (see e.g.,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (forexample, metallothionein promoter) or from mammalian viruses (forexample, the retrovirus long terminal repeat; the adenovirus latepromoter; the vaccinia virus 7.5K promoter) can be used. Promotersproduced by recombinant DNA or synthetic techniques may also be used toprovide for transcription of the nucleic acid sequences.

Protein purification: the fusion polypeptides disclosed herein can bepurified (and/or synthesized) by any of the means known in the art (see,e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol. 185,Academic Press, San Diego (1990); and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York (1982). Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least 60%, 70%, 80%,90%, 95% or 98% pure. Thus, in one specific, non-limiting example, asubstantially purified protein is 90% free of other proteins or cellularcomponents.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified nucleicacid is one in which the nucleic acid is more enriched than the nucleicacid in its natural environment within a cell. Similarly, a purifiedpeptide preparation is one in which the peptide or protein is moreenriched than the peptide or protein is in its natural environmentwithin a cell. In one embodiment, a preparation is purified such thatthe protein or peptide represents at least 50% (such as, but not limitedto, 70%, 80%, 90%, 95%, 98% or 99%) of the total peptide or proteincontent of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence,for example, a polynucleotide encoding a fusion protein. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques.

Sequence identity: The similarity between amino acid (andpolynucleotide) sequences is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity); the higher the percentage, the more similar are theprimary structures of the two sequences. In general, the more similarthe primary structures of two amino acid sequences, the more similar arethe higher order structures resulting from folding and assembly.However, the converse is not necessarily true, and polypeptides with lowsequence identity at the amino acid level can nonetheless have highlysimilar tertiary and quaternary structures. For example, NSP2 homologswith little sequence identity (for example, less than 50% sequenceidentity, or even less than 30%, or less than 20% sequence identity)share similar higher order structure and assembly properties, such thateven distantly related NSP2 proteins assemble into multimeric ringstructures as described herein.

Methods of determining sequence identity are well known in the art.Various programs and alignment algorithms are described in: Smith andWaterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol.Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Another indicia of sequence similarity between two nucleic acids is theability to hybridize. The more similar are the sequences of the twonucleic acids, the more stringent the conditions at which they willhybridize. The stringency of hybridization conditions aresequence-dependent and are different under different environmentalparameters. Thus, hybridization conditions resulting in particulardegrees of stringency will vary depending upon the nature of thehybridization method of choice and the composition and length of thehybridizing nucleic acid sequences. Generally, the temperature ofhybridization and the ionic strength (especially the Na⁺ and/or Mg⁺concentration) of the hybridization buffer will determine the stringencyof hybridization, though wash times also influence stringency.Generally, stringent conditions are selected to be about 5° C. to 20° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Conditions for nucleic acidhybridization and calculation of stringencies can be found, for example,in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Tijssen,Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic AcidPreparation, Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Ltd., NY, N.Y., 1993 and Ausubel et al. ShortProtocols in Molecular Biology, 4^(th) ed., John Wiley & Sons, Inc.,1999.

For purposes of the present disclosure, “stringent conditions” encompassconditions under which hybridization will only occur if there is lessthan 25% mismatch between the hybridization molecule and the targetsequence. “Stringent conditions” may be broken down into particularlevels of stringency for more precise definition. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 25% sequence mismatch will not hybridize; conditions of“medium stringency” are those under which molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichsequences with more than 6% mismatch will not hybridize. In contrastnucleic acids that hybridize under “low stringency conditions includethose with much less sequence identity, or with sequence identity overonly short subsequences of the nucleic acid.

For example, a specific example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Therapeutically active polypeptide: An agent, such as an epitope of avirus or other pathogen that causes induction of an immune response, asmeasured by clinical response (for example increase in a population ofimmune cells, increased cytolytic activity against the epitope).Therapeutically active molecules can also be made from nucleic acids.Examples of a nucleic acid based therapeutically active molecule is anucleic acid sequence that encodes an epitope of a protein of a virus orother pathogen, wherein the nucleic acid sequence is operatively linkedto a control element such as a promoter.

Therapeutically Effective Amount: An amount of a composition that alone,or together with an additional therapeutic agent(s) (for examplenucleoside/nucleotide reverse transcriptase inhibitors, a non-nucleosidereverse transcriptase inhibitors, protease inhibitors, fusion/entryinhibitors or integrase inhibitors) induces the desired response (e.g.,inhibition of HIV infection or replication). In one example, a desiredresponse is to inhibit HIV replication in a cell to which the therapy isadministered. HIV replication does not need to be completely eliminatedfor the composition to be effective. For example, a composition candecrease HIV replication by a desired amount, for example by at least10%, at least 20%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, or even at least 100%(elimination of HIV), as compared to HIV replication in the absence ofthe composition.

In another example, a desired response is to inhibit HIV infection. TheHIV infected cells do not need to be completely eliminated for thecomposition to be effective. For example, a composition can decrease thenumber of HIV infected cells by a desired amount, for example by atleast 10%, at least 20%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination of detectable HIV infected cells), as compared to thenumber of HIV infected cells in the absence of the composition.

A therapeutically effective amount of a composition including variantHBsAgs, can be administered in a single dose, or in several doses, forexample daily, during a course of treatment. However, thetherapeutically effective amount can depend on the subject beingtreated, the severity and type of the condition being treated, and themanner of administration. For example, a therapeutically effectiveamount of such agent can vary from about 1 μg-10 mg per 70 kg bodyweight if administered intravenously.

Tissue: A plurality of functionally related cells. A tissue can be asuspension, a semi-solid, or solid. Tissue includes cells collected froma subject.

Transformed or Transfected: A transformed cell is a cell into which anucleic acid molecule has been introduced by molecular biologytechniques. As used herein, the term introduction or transformationencompasses all techniques by which a nucleic acid molecule might beintroduced into such a cell, including transfection with viral vectors,transformation with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration.

Transmembrane domain or region of gp41: A region or domain of gp41 thatis immediately C-terminal to the membrane proximal region of gp41. Anexample of a transmembrane domain is provided in SEQ ID NO: 25.

Treating a disease: “Treatment” refers to a therapeutic interventionthat ameliorates a sign or symptom of a disease or pathologicalcondition, such as a sign or symptom of HIV. Treatment can also induceremission or cure of a condition, such as elimination of detectable HIVinfected cells. In particular examples, treatment includes preventing adisease, for example by inhibiting the full development of a disease,such as HIV, by inhibiting HIV replication or infection or thedevelopment of AIDS. Prevention of a disease does not require a totalabsence of disease. For example, a decrease of at least 50% can besufficient.

Vaccine: A vaccine is a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective immune response. Typically, avaccine elicits an antigen-specific immune response to an antigen of apathogen, for example, a bacterial or viral pathogen, or to a cellularconstituent correlated with a pathological condition. A vaccine mayinclude a polynucleotide, a peptide or polypeptide, a virus, a bacteria,a cell or one or more cellular constituents. In some cases, the virus,bacteria or cell may be inactivated or attenuated to prevent or reducethe likelihood of infection, while maintaining the immunogenicity of thevaccine constituent.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat, and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle. Avirus may subvert the host cells' normal functions, causing the cell tobehave in a manner determined by the virus. For example, a viralinfection may result in a cell producing a cytokine, or responding to acytokine, when the uninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV). HIV-1 is a retrovirus that causesimmunosuppression in humans (HIV disease), and leads to a diseasecomplex known as AIDS. “HIV infection” refers to the process in whichHIV enters macrophages and CD4+ T cells by the adsorption ofglycoproteins on its surface to receptors on the target cell followed byfusion of the viral envelope with the cell membrane and the release ofthe HIV capsid into the cell. “HIV disease” refers to a well-recognizedconstellation of signs and symptoms (including the development ofopportunistic infections) in persons who are infected by an HIV virus,as determined by antibody or western blot studies. Laboratory findingsassociated with this disease are a progressive decline in T cells.

Virus-like particle or VLP: A nonreplicating, viral shell, derived fromany of several viruses. VLPs are generally composed of one or more viralproteins, such as, but not limited to, those proteins referred to ascapsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art. The presence of VLPs following recombinant expressionof viral proteins can be detected using conventional techniques known inthe art, such as by electron microscopy, biophysical characterization,and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; Hagensee et al. (1994) J. Virol. 68:4503-4505. Forexample, VLPs can be isolated by density gradient centrifugation and/oridentified by characteristic density banding. Alternatively,cryoelectron microscopy can be performed on vitrified aqueous samples ofthe VLP preparation in question, and images recorded under appropriateexposure conditions.

II. Description of Several Embodiments

Historically, viral vaccines have been live-attenuated or chemicallyinactivated forms of the virus. However, this approach has limitedutility when used for HIV. Recombinant HBsAg-gp120 has been used topresent approximately amino acids 1-500 of gp120. However, thepresentation of gp120 in this form has not successfully been used toproduce neutralizing antibodies.

Human monoclonal antibodies to the MPR of gp41 can neutralize verybroadly, including virtually all HIV isolates in North America andWestern Europe. Therefore, many researchers have tried to express theMPR determinant in ways that could elicit neutralizing antibodiessimilar to the two monoclonals specific for this site which wereobtained from infected humans. However, immunization has failed toelicit these antibodies. Without being bound by a particular theory, theinability to elicit neutralizing antibodies may be due to improperorientation of the MPR on the lipid surface.

The inventors have developed isolated immunogens including a variantHBsAg. HBsAg was selected as a carrier protein because it spontaneouslyassembles into virus-like particles. These particles have a lipid layer,and HBsAg embeds itself in the lipid layer, which it spans four times.The disclosed variant HBsAgs include a HBsAg with one or moretransmembrane domains of the HBsAg replaced with a gp41 antigenicinsert. The gp41 antigenic insert includes the MPR of gp41 and atransmembrane region of gp41. The replacement of a membrane spanningdomain of HBsAg, such as the first and/or third transmembrane domains,with a membrane spanning domain of gp41 anchors gp41 into HBsAgparticles in virtually the identical orientation as on HIV virions andcorrectly orients the nearby MPR on the lipid layer. The disclosed HBsAgvariants are readily recognized by human monoclonal antibodies, 2F5,4E10, and Z13e, which bind and neutralize a broad spectrum of HIVisolates. Thus, the disclosed particles display theneutralization-sensitive MPR in association with a lipid layer, whilepresenting it at the most immunogenic site on HBsAg. The results showthat HBsAg is remarkably flexible in particle formation, and it canassemble particles, even with the new membrane spanning domain in place.

Disclosed herein is the use of the immunogenic HBsAg particulateplatform to array epitopes from the conserved, neutralization-sensitiveMPR of HIV-1, and the use of this platform to induce an immune responseto HIV-1 using specific antigenic epitopes of gp41. Specifically, it isdisclosed herein that the HBsAg can be used as a carrier, for example ina multi-array presentation of the antigenic components of the HIVenvelope protein (env), such as to induce an immune response to highlyconserved, hydrophobic 2F5 and 4E10 neutralizing determinants from gp41.In addition, the use of the HBsAg platform allows presentation of theMPR as an immunogen in an appropriate lipid context.

A. Isolated Immunogens with Variant HBsAgs

Isolated immunogens including variant HBsAgs are disclosed. In anexample, a variant HBsAg includes an HBsAg with one or moretransmembrane domains of the HBsAg replaced with a gp41 transmembranespanning domain and/or one or more gp41 MPRs. The replacement of amembrane spanning domain of HBsAg with a membrane spanning domain ofgp41 anchors gp41 into HBsAg in virtually the identical orientation ason HIV virions and correctly orients the nearby MPR on the lipid layer.Thus, the disclosed variant HBsAgs display the neutralization-sensitiveMPR in association with a lipid layer, while presenting it at the mostimmunogenic site on HBsAg.

Suitable amino acid sequences for HBsAg are known in the art, and aredisclosed, for example, in PCT Publication No. WO 2002/079217, which isincorporated herein by reference. Additional sequences for hepatitis Bsurface antigen can be found, for example, in PCT Publication No.2004/113369 and PCT Publication No. WO 2004/09849. An exemplary HBsAgamino acid sequence, and the sequence of a nucleic acid encoding HBsAg,is shown in Berkower et al., Virology 321: 74-86, 2004, which isincorporated herein by reference in its entirety. An amino acid sequenceof an exemplary HBsAg is set forth as follows:

(SEQ ID NO: 31) EFITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCISIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG.

By itself, HBsAg assembles into approximately 22 nm virus-likeparticles. When expressed together with an HIV-1 antigenic epitope, theHBsAg fusion proteins assemble spontaneously and efficiently intovirus-like particles (see Berkower et al., Virology 321: 75-86, 2004,which is incorporated herein by reference). Without being bound bytheory, the multimeric form expresses the one or more antigenic epitopesat the lipid-water interface. These epitopes can be used to induce animmune response, such as to induce the production of neutralizingantibodies.

The preparation of hepatitis B surface antigen (HBsAg) is welldocumented. See, for example, Harford et al. (1983) Develop. Biol.Standard 54:125; Greg et al. (1987) Biotechnology 5:479; EP-A-0 226 846;and EP-A-0 299 108.

Fragments and variants of HBsAgs as disclosed herein are fragments andvariants that retain the ability to spontaneously assemble intovirus-like particles. By “fragment” of a HBsAg is intended a portion ofa nucleotide sequence encoding a HBsAg, or a portion of the amino acidsequence of the protein. By “homologue” or “variant” is intended anucleotide or amino acid sequence sufficiently identical to thereference nucleotide or amino acid sequence, respectively.

It is recognized that the gene or cDNA encoding a polypeptide can beconsiderably mutated without materially altering one or more of thepolypeptide's functions. The genetic code is well known to bedegenerate, and thus different codons encode the same amino acids. Evenwhere an amino acid substitution is introduced, the mutation can beconservative and have no material impact on the essential functions of aprotein (see Stryer, Biochemistry 4th Ed., W. Freeman & Co., New York,N.Y., 1995). Part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. Sequence variants of aprotein, such as a 5′ or 3′ variant, can retain the full function of anentire protein. Moreover, insertions or additions can be made in thepolypeptide chain for example, adding epitope tags, without impairing oreliminating its functions (Ausubel et al., Current Protocols inMolecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998).Specific substitutions include replacing one or more transmembranespanning domains of HBsAg with a gp41 transmembrane spanning domain,such replacing the first domain and/or third domain of HBsAg with a gp41transmembrane spanning domain. Other modifications that can be madewithout materially impairing one or more functions of a polypeptideinclude, for example, in vivo or in vitro chemical and biochemicalmodifications or the incorporation of unusual amino acids. Suchmodifications include, for example, acetylation, carboxylation,phosphorylation, glycosylation, ubiquination, labeling, such as withradionucleides, and various enzymatic modifications, as will be readilyappreciated by those well skilled in the art. A variety of methods forlabeling polypeptides and labels useful for such purposes is well knownin the art, and includes radioactive isotopes such as ³²P, ligands thatbind to or are bound by labeled specific binding partners (such asantibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands.

Functional fragments and variants of HBsAg include those fragments andvariants that are encoded by nucleotide sequences that retain theability to spontaneously assemble into virus-like particles. Functionalfragments and variants can be of varying length. For example, a fragmentmay consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 ormore, or 200 or more amino acid residues of a HBsAg amino acid sequence.

A functional fragment or variant of HBsAg is defined herein as apolypeptide that is capable of spontaneously assembling into virus-likeparticles and/or self-aggregating into stable multimers. This includes,for example, any polypeptide six or more amino acid residues in lengththat is capable of spontaneously assembling into virus-like particles.Methods to assay for virus-like particle formation are well known in theart (see, for example, Berkower et al. (2004) Virology 321:75-86, hereinincorporated by reference in its entirety).

“Homologues” or “variants” of a hepatitis B surface antigen are encodedby a nucleotide sequence sufficiently identical to a nucleotide sequenceof hepatitis B surface antigen, examples of which are described above.By “sufficiently identical” is intended an amino acid or nucleotidesequence that has at least about 60% or 65% sequence identity, about 70%or 75% sequence identity, about 80% or 85% sequence identity, about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over itsfull length as compared to a reference sequence, for example using theNCBI Blast 2.0 gapped BLAST set to default parameters. Alignment mayalso be performed manually by inspection. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). In one embodiment, the HBsAg protein is atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%identical to the polypeptide of SEQ ID NO: 31.

One or more conservative amino acid modifications can be made in theHBsAg amino acid sequence, whether an addition, deletion ormodification, that does not substantially alter the 3-dimensionalstructure of the polypeptide. For example, a conservative amino acidsubstitution does not affect the ability of the HBsAg polypeptide toself-aggregate into stable multimers. HBsAg proteins having deletions ofa small number of amino acids, for example, less than about 10% (such asless than about 8%, or less than about 5%, or less than about 2%, orless than about 1%) of the total number of amino acids in the wild typeHBsAg protein can also be included in the fusion proteins describedherein. The deletion may be a terminal deletion, or an internaldeletion, so long as the deletion does not substantially affect thestructure or aggregation of the fusion protein.

In certain embodiments, a variant HBsAg can include a linker sequence.This peptide is a short amino acid sequence providing a flexible linkerthat permits attachment of an antigenic polypeptide without disruptionof the structure, aggregation (multimerization) or activity of theself-aggregating polypeptide component. Typically, a linear linkingpeptide consists of between two and 25 amino acids. Usually, the linearlinking peptide is between two and 15 amino acids in length. In oneexample, the linker polypeptide is two to three amino acids in length,such as a serine and an arginine, or two serine residues and an arginineresidue, or two arginine residues and a serine residue.

In other examples, the linear linking peptide can be a short sequence ofalternating glycines and prolines, such as the amino acid sequenceglycine-proline-glycine-proline. A linking peptide can also consist ofone or more repeats of the sequence glycine-glycine-serine.Alternatively, the linear linking peptide can be somewhat longer, suchas the glycine(4)-serine spacer described by Chaudhary et al., Nature339:394-397, 1989.

Directly or indirectly adjacent to the remaining end of the linearlinking peptide (that is, the end of the linear linking peptide notattached to the self-aggregating polypeptide component of the fusionprotein) is a polypeptide sequence including at least one antigenicepitope of HIV-1, such as an epitope of gp41, such as at least oneantigenic epitope of the membrane proximal region. The antigenicpolypeptide can be a short peptide sequence including a single epitope.For example the antigenic polypeptide can be a sequence of amino acidsas short as eight or nine amino acids, sufficient in length to providean antigenic epitope in the context of presentation by a cellularantigen presenting complex, such as the major histocompatibility complex(MHC). The antigenic polypeptide can also be of sufficient in length toinduce antibodies, such as neutralizing antibodies. Larger peptides, inexcess of 10 amino acids, 20 amino acids or 30 amino acids are alsosuitable antigenic polypeptides, as are much larger polypeptidesprovided that the antigenic polypeptide does not disrupt the structureor aggregation of the HBsAg polypeptide component.

In some examples, the variant HBsAg includes one or more epitopes of theenvelope protein of HIV-1, and is about 20 to about 200 amino acids inlength, such as about 25 to about 150 amino acids in length, such asabout 25 to about 100 amino acids in length. In several additionalexamples, the antigenic polypeptide includes one or more antigenicepitopes of HIV-1 gp41, such as the membrane proximal region (MPR) ofgp41.

Exemplary sequences for HIV-1, as well as the amino acid sequence forfull-length gp41 can be found on Genbank, EMBL and SwissProt websites.Exemplary non-limiting sequence information can be found for example, asSwissProt Accession No. PO4578, (includes gp41 and gp120, initial entryAug. 13, 1987, last modified on Jul. 15, 1999); Genbank Accession No.HIVHXB2CG (full length HIV-1, including RNA sequence and encodedproteins, Oct. 21, 2002); Genbank Accession No. CAD23678 (gp41, Apr. 15,2005); Genbank Accession No. AAF69493 (Oct. 2, 2000, gp120); GenbankAccession No. CAA65369 (Apr. 18, 2005); all of which are incorporatedherein by reference. Similar information is available for HIV-2.

Suitable Env proteins are known in the art and include, for example,gp160, gp120, gp41, and gp140. Any clade of HIV is appropriate forantigen selection, including HIV clades A, B, C, and the like. HIV Gag,Pol, Nef and/or Env proteins from HIV clades A, B, C, as well as nucleicacid sequences encoding such proteins and methods for the manipulationand insertion of such nucleic acid sequences into vectors, are known(see, for example, HIV Sequence Compendium, Division of AIDS, NationalInstitute of Allergy and Infectious Diseases, 2003, HIV SequenceDatabase (on the world wide web athiv-web.lanl.gov/content/hiv-db/mainpage.html), Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Association. ExemplaryEnv polypeptides, for example, corresponding to clades A, B and C arerepresented by the sequences of Genbank® Accession Nos. U08794, K03455and AF286227, respectively.

Variant HBsAgs can form a self-aggregating multimeric ring structureupon expression in a host cell. Similarly, the variant HBsAgs canassemble spontaneously (self-aggregate) when placed in suspension in asolution of physiological pH (for example, a pH of about 7.0 to 7.6).Thus, in the present disclosure, wherever a single or monomeric variantHBsAg is disclosed, polymeric forms are also considered to be described.

In some embodiments, an isolated immunogen includes a variant HBsAg withone or more transmembrane domains of the HBsAg replaced with a gp41antigenic insert. The gp41 antigenic insert can include (a) an antigenicpolypeptide fragment of gp41 and (b) a transmembrane domain of gp41. Inan example, the gp41 antigenic insert includes (a) an antigenicpolypeptide fragment, such as an antigenic polypeptide fragment with theamino acid sequence set forth in SEQ ID NO:1 and is between 28 and 150amino acids in length and (b) a transmembrane spanning gp41 region, suchas a transmembrane spanning gp41 region with the amino acid sequence setforth in SEQ ID NO: 25 (in which wherein X₁, X₂, X₃, and X₄ are anyamino acid and X₅, X₆, and X₇ are any hydrophobic amino acid) and isbetween 22 and 40 amino acids in length.

In one example, the antigenic polypeptide includes the amino acidsequence of NEX₁X₂LLX₃LDKWASLWNWFDITNWLWYIX₄ (SEQ ID NO: 1). In thissequence, X₁, X₂ X₃, and X₄ are any amino acid. The antigenic epitopecan include repeats of this sequence, such as one to five copies of SEQID NO: 1. As noted above, the antigenic peptide includes one or moreepitopes of the envelope protein of HIV-1, and, including SEQ ID NO: 1,can be from about 28 to about 200 amino acids in length, such from about28 to about 150 amino acids in length, such as from about 28 to about140 amino acids in length.

In several examples, the antigenic polypeptide includes one or more ofthe amino acid sequences set forth below:

a)  SEQ ID NO: 2 (NEQELLALDKWASLWNWFDITNWLWYIK); b) SEQ ID NO: 3(NEQDLLALDKWASLWNWFDITNWLWYIK); c) SEQ ID NO: 4(NEQDLLALDKWANLWNWFDISNWLWYIK); d) SEQ ID NO: 5(NEQDLLALDKWANLWNWFNITNWLWYIR); e) SEQ ID NO: 6 (NEQELLELDKWASLWNWFDITNWLWYIK); f)  SEQ ID NO: 7(NEKDLLALDSWKNLWNWFDITNWLWYIK); g)  SEQ ID NO: 8(NEQDLLALDSWENLWNWFDITNWLWYIK); h)  SEQ ID NO: 9 (NEQELLELDKWASLWNWFSITQWLWYIK); i)  SEQ ID NO: 10(NEQELLALDKWASLWNWFDISNWLWYIK); j)  SEQ ID NO: 11(NEQDLLALDKWDNLWSWFTITNWLWYIK); k)  SEQ ID NO: 12(NEQDLLALDKWASLWNWFDITKWLWYIK); l)  SEQ ID NO: 13(NEQDLLALDKWASLWNWFSITNWLWYIK); m)  SEQ ID NO: 14(NEKDLLELDKWASLWNWFDITNWLWYIK); n)  SEQ ID NO: 15(NEQEILALDKWASLWNWFDISKWLWYIK); o)  SEQ ID NO: 16(NEQDLLALDKWANLWNWFNISNWLWYIK); p)  SEQ ID NO: 17(NEQDLLALDKWASLWSWFDISNWLWYIK); q)  SEQ ID NO: 18(NEKDLLALDSWKNLWSWFDITNWLWYIK); r)  SEQ ID NO: 19(NEQELLQLDKWASLWNWFSITNWLWYIK); s)  SEQ ID NO: 20(NEQDLLALDKWASLWNWFDISNWLWYIK); t)  SEQ ID NO: 21(NEQELLALDKWASLWNWFDISNWLWYIR); u)  SEQ ID NO: 22(NEQELLELDKWASLWNWFNITNWLWYIK);  or v)  SEQ ID NO: 23 (NEKELLELDKWASLWNWFDITNWLWYI) as in the TM 12 construct: w)SEQ ID NO: 24 (NEKELLELDKWASLWNWFDITNWLWYIR) as in the TM14, TM16 or TM20 construct; x)  SEQ ID NO: 60 (NEKELLELDKWASLW) as repeated four times in the TM32F construct;  or y) SEQ ID NO: 61   (NEKELLELDKWASLWNWFDITNWLW) as in the TM34 construct . .

The antigenic polypeptide can include one of the amino acid sequencesset forth as SEQ ID NOs: 2-24 or 60, 61. A single copy of one of SEQ IDNOs: 2-24 or 60, 61 can be included as the antigenic polypeptide.Alternatively, multiple copies of one of SEQ ID NOs: 2-24, 60 or 61 canbe included as the antigenic polypeptide. Thus, one, two, three, four orfive copies or more of one of the amino acid sequences set forth as SEQID NOs: 2-24, 60 or 61 can be included as the antigenic polypeptide.

In additional embodiments, more than one of these sequences can beincluded in the antigenic polypeptide. Thus, in several examples, two,three, four, five or more of the amino acid sequences set forth as SEQID NOs: 2-24, 60 or 61 can be included as the antigenic polypeptide.Each amino acid sequence included in the antigenic polypeptide can bepresent only a single time, or can be repeated.

In some embodiments, the transmembrane spanning gp41 region includes theamino acid sequence set forth in SEQ ID NO: 25. In this sequence, X₁,X₂, X₃, and X₄ are any amino acid and X₅, X₆, and X₇ and thetransmembrane spanning gp41 region is between 22 and 40 amino acids inlength. In several examples, the antigenic polypeptide includes one ormore of the amino acid sequences set forth below:

a)  SEQ ID NO: 26  (IFIMIVGGLIGLRIVFTVLSIV) b) SEQ ID NO: 27(LFIMIVGGLIGLRIVFTALSIV);  or c) SEQ ID NO: 28 (IFIMIVGGLVGLRIVFTALSIV)

The HBsAg variants can include one or more transmembrane spanningdomains that include one of the amino acid sequences set forth as SEQ IDNOs: 26-28. A single gp41 transmembrane can be included in the variantHBsAg. Alternatively, multiple gp41 transmembrane domains with aminoacid sequences set forth as SEQ ID NOs: 26-28 can be included within thevariant HBsAg. Thus, one, two, three, four or five gp41 transmembranedomains with one of the amino acid sequences set forth as SEQ ID NOs:26-28 can be included in the variant HBsAg.

In one particular embodiment, an isolated immunogen includes a variantHBsAg in which the first transmembrane spanning domain of the HBsAg isreplaced by a gp41 antigenic insert. For example, the gp41 antigenicinsert replaces at least the first 29 amino acid residues of SEQ IDNO:31, for example amino acid residues 1-35 of SEQ ID NO: 31. In anotherexample, the gp41 antigenic insert replaces amino acid residues 1-32 ofSEQ ID NO: 31. In yet another example, the gp41 antigenic insertreplaces amino acid residues 1-29 of SEQ ID NO: 31. In a particularexample, an isolated immunogen includes a variant HBsAg in which thefirst transmembrane spanning domain of the HBsAg is replaced by a gp41antigenic insert that has the amino acid sequence set forth as SEQ IDNO: 29.

In another particular embodiment, an isolated immunogen includes avariant HBsAg in which the third transmembrane spanning domain of theHBsAg is replaced by a gp41 antigenic insert. For example, the gp41antigenic insert replaces at least 29 amino acids residues of SEQ ID NO:31, for example amino acid residues 150-190 of SEQ ID NO: 31. In anotherexample, the gp41 antigenic insert replaces amino acid residues 153-187of SEQ ID NO: 31. In yet another example, the gp41 antigenic insertreplaces amino acid residues 156-185 of SEQ ID NO: 31. In a particularexample, an isolated immunogen including a variant HBsAg in which thethird transmembrane spanning domain of the HBsAg is replaced by a gp41antigenic insert has the amino acid sequence set forth as SEQ ID NO: 57or an amino acid sequence that is at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence similarity, such as 80%,82%, 85%, 87%, 90%, 93%, 95% or 98% sequence similarity with suchsequence.

In an even more particular embodiment, an isolated immunogen includes avariant HBsAg in which more than one transmembrane spanning domain ofHBsAg has been replaced with an antigenic insert. In one example, anisolated immunogen includes a variant HBsAg in which the first and thethird transmembrane spanning domains of the HBsAg are replaced by a gp41antigenic insert. For example, the gp41 antigenic insert replaces aminoacid residues 1-35 and 150-190 of SEQ ID NO: 31. In another example, thegp41 antigenic insert replaces amino acid residues 1-32 and 153-187 ofSEQ ID NO: 31. In yet another example, the gp41 antigenic insertreplaces amino acid residues 1-29 and 156-185 of SEQ ID NO: 31.

In one example of an isolated immunogen, the first transmembrane domainof HBsAg is replaced with the MPR and transmembrane domain of gp41 andhas the amino acid sequence set forth as:

(SEQ ID NO: 29; TM16) MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASLWNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG.

In one example of an isolated immunogen, the third transmembrane domainof HBsAg is replaced with the MPR and transmembrane domain of gp41 andsuch immunogen has the amino acid sequence set forth as:

(SEQ ID NO: 57; TM20) MKTIIALSYIFCLVFAQDLPGNDNNSEFITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCININEKELLELDKWASLWNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIVVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG.

In an example, an isolated immunogen is provided in which the firsttransmembrane domain and third domain of HBsAg is each replaced with theMPR and transmembrane domain of gp41 and has the amino acid sequence setforth as:

(SEQ ID NO: 58; TM16 + TM20)..................................................MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASLWNWFDITN..................................................WLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQ..................................................NSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQ..................................................GMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCIP..................................................INEKELLELDKWASLWNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSI.............................................VVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG.

In one example, an isolated immunogen is provided in which the secondand third domain of HBsAg is replaced with a MPR and has the amino acidsequence set forth as:

(SEQ ID NO: 56; TM34) MKTIIALSYIFCLVFAQDLPGNDNNSEFITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWNWFDITNWLWSSLWAIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG 

In one example, an isolated immunogen is provided in which the firstdomain of HBsAg is replaced with a MPR and transmembrane domain of gp41and the domain between the second and third domain of HBsAg is replacedwith a MPR and has the amino acid sequence set forth as:

(SEQ ID NO: 62; TM16 + TM34)MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASLWNWFDITN  WLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQ  NSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWNWFDITNWLWSSLWAIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG 

In one example, an isolated immunogen is provided in which four MPRs areinserted between a second and third domain in the HBsAg construct andhas the amino acid sequence set forth as:

(SEQ ID NO: 63; TM32F)MKTIIALSYIFCLVFAQDLPGNDNNSEFITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWAINEKELLELDKWASLWAINEKELLELDKWASLWAINEKELLELDKWASLWAIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIP  LLPIFFCLWVYIG

In one example, an isolated immunogen is provided in which the firstdomain of HBsAg is replaced with a MPR and transmembrane domain of gp41and four additional MPRs are inserted between a second and third domainin the HBsAg construct and the construct has a sequence set forth as:

(SEQ ID NO: 64; TM16 + TM32F)MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASLWNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWAINEKELLELDKWASLWAINEKELLELDKWASLWAINEKELLELDKWASLWAIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG.

The variant HBsAg can optionally include additional elements, such as aleader sequence (such as a leader sequence from influenza hemagglutinin,see, for example, SEQ ID NO: 30 as included within variant HBsAgs withamino acid sequences set forth by SEQ ID NOs: 29, 56-58 and 62-64) or asuitable T cell epitope. Generally, a T cell epitope is about eight toabout ten amino acids in length, such as about nine amino acid inlength, and binds major histocompatibility complex (MHC), such as HLA 2,for example, HLA 2.2. Examples of suitable T cell epitopes include, butare not limited to, ASLWNWFNITNWLWY (SEQ ID NO: 32) and IKLFIMIVGGLVGLR(SEQ ID NO: 33).

The variant HBsAg may also include a CAAX (SEQ ID NO: 34) sequence, forisoprenyl addition in vivo. In this sequence, C is cysteine, A is analiphatic amino acid and X is any amino acid. The X residue determineswhich isoprenoid will be added to the cysteine. When X is a methionineor serine, the farnesyl-transferase transfers a farnesyl, and when X isa leucine or isoleucine, the geranygeranyl-transferase I, ageranylgeranyl group. In general, aliphatic amino acids have proteinside chains containing only carbon or hydrogen atoms. Aliphatic aminoacids include proline (P), glycine (G), alanine (A), valine (V), leucine(L), and isoleucine (I), presented in order from less hydrophobic tomore hydrophobic. Although methionine has a sulphur atom in itsside-chain, it is largely non-reactive, meaning that methionineeffectively substitutes well with the true aliphatic amino acids.

B. Polynucleotides Encoding Variant HBsAgs

Nucleic acids encoding the variant HBsAgs described herein are alsoprovided. These nucleic acids include deoxyribonucleotides (DNA, cDNA)or ribodeoxynucleotides (RNA) sequences, or modified forms of eithernucleotide, which encode the variant HBsAgs described herein. The termincludes single and double stranded forms of DNA and/or RNA. The nucleicacids can be operably linked to expression control sequences, such as,but not limited to, a promoter.

The nucleic acids that encode the variant HBsAgs disclosed hereininclude a polynucleotide sequence that encodes a variant HBsAgsincluding a HBsAg with one or more MPRs and/or one or more transmembranedomains of the HBsAg replaced with a gp41 antigenic insert.

In one example, nucleic acids that encode a variant HBsAg in which athird transmembrane domain of HBsAg is replaced with a gp41 antigenicinsert has the nucleotide sequence set forth as

(SEQ ID NO: 59) GGTACCGTCGACAGCAAAAGCAGGGGATAATTCTATTAACCATGAAGACTATCATTGCTTCCATGGCAGCTGTCGTTTTCGTCCCCTATTAAGATAATTGGTACTTCTGATAGTAACGAATGAGCTACATTTTCTGTCTGGTTTTCGCCCAAGACCTTCCAGGAAATGACAACAACAGCGACTCGATGTAAAAGACAGACCAAAAGCGGGTTCTGGAAGGTCCTTTACTGTTGTTGTCGCAATTCATCACCTCCGGCTTCCTGGGCCCCCTGCTGGTCCTGCAGGCCGGGTTCTTCCTGCTTAAGTAGTGGAGGCCGAAGGACCCGGGGGACGACCAGGACGTCCGGCCCAAGAAGGACGTGACCCGCATCCTCACCATCCCCCAGTCCCTGGACTCGTGGTGGACCTCCCTCAACTTTCACTGGGCGTAGGAGTGGTAGGGGGTCAGGGACCTGAGCACCACCTGGAGGGAGTTGAAAGTGGGGGGCTCCCCCGTGTGTCTGGGCCAGAACTCCCAGTCCCCCACCTCCAACCACTCCCACCCCCCGAGGGGGCACACAGACCCGGTCTTGAGGGTCAGGGGGTGGAGGTTGGTGAGGGCCACCTCCTGCCCCCCCATCTGCCCCGGCTACCGCTGGATGTGCCTGCGCCGCTTCATCAGGTGGAGGACGGGGGGGTAGACGGGGCCGATGGCGACCTACACGGACGCGGCGAAGTAGTTCTTCCTGTTCATCCTGCTGCTGTGCCTGATCTTCCTGCTGGTGCTGCTGGACTACCAGGAGAAGGACAAGTAGGACGACGACACGGACTAGAAGGACGACCACGACGACCTGATGGTCCGCATGCTGCCCGTGTGCCCCCTGATCCCCGGCTCCACCACCACCTCCACCGGCCCCTGCACGTACGACGGGCACACGGGGGACTAGGGGCCGAGGTGGTGGTGGAGGTGGCCGGGGACGTAGACCTGCACCACCCCCGCCCAGGGCAACTCCAAGTTCCCCTCCTGCTGCTGCACCAAGCTCTGGACGTGGTGGGGGCGGGTCCCGTTGAGGTTCAAGGGGAGGACGACGACGTGGTTCGCCACCGACGGCAACTGCACCTGCATCAATATTAATGAAAAAGAATTATTGGAATTGGATAGGTGGCTGCCGTTGACGTGGACGTAGTTATAATTACTTTTTCTTAATAACCTTAACCTATAATGGGCAAGTTTGTGGAATTGGTTTGACATAACAAACTGGCTGTGGTATATAAGATTATTTACCCGTTCAAACACCTTAACCAAACTGTATTGTTTGACCGACACCATATATTCTAATATCATAATGATAGTAGGAGGCTTGATAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGAGTATTACTATCATCCTCCGAACTATCCAAATTCTTATCAAAAACGACATGAAAGATATCTAGTGGGCCTGTCCCCCACCGTGTGGCTGTCCGCCATCTGGATGATGTGGTACTGGGGCCATCACCCGGACAGGGGGTGGCACACCGACAGGCGGTAGACCTACTACACCATGACCCCGGCCTCCCTGTACTCCATCGTGTCCCCCTTCATCCCCCTGCTGCCCATCTTCTTCTGCCTGTGGAGGGACATGAGGTAGCACAGGGGGAAGTAGGGGGACGACGGGTAGAAGAAGACGGACAGGGTGTACATCTGACTAGTGAGCTCCCCACATGTAGACTGATCACTCGAG.

The variant HBsAgs and the polynucleotides encoding them describedherein can be used to produce pharmaceutical compositions, includingcompositions suitable for prophylactic and/or therapeuticadministration. These compositions can be used to induce an immuneresponse to HIV, such as a protective immune response. However, thecompositions can also be used in various assays, such as in assaysdesigned to detect an HIV-1 infection.

Methods and plasmid vectors for producing the polynucleotides encodingvariant HBsAgs and for expressing these polynucleotides in bacterial andeukaryotic cells are well known in the art, and specific methods aredescribed in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,Ch. 17, CSHL, New York, 1989). Such variant HBsAgs may be made in largeamounts, are easy to purify, and can be used to elicit an immuneresponse, including an antibody response and/or a T cell response.Native proteins can be produced in bacteria by placing a strong,regulated promoter and an efficient ribosome-binding site upstream ofthe cloned gene. If low levels of protein are produced, additional stepsmay be taken to increase protein production; if high levels of proteinare produced, purification is relatively easy. Suitable methods arepresented in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989) and are well known in the art. Often, proteinsexpressed at high levels are found in insoluble inclusion bodies.Methods for extracting proteins from these aggregates are described bySambrook et al. (In Molecular Cloning: A Laboratory Manual, Ch. 17,CSHL, New York, 1989). Proteins, including variant HBsAgs, may beisolated from protein gels, lyophilized, ground into a powder and usedas an antigen.

Vector systems suitable for the expression of polynucleotides encodingvariant HBsAgs include, in addition to the specific vectors described inthe examples, the pUR series of vectors (Ruther and Muller-Hill, EMBO J.2:1791, 1983), pEX1-3 (Stanley and Luzio, EMBO J. 3:1429, 1984) andpMR100 (Gray et al., Proc. Natl. Acad. Sci. USA 79:6598, 1982). Vectorssuitable for the production of intact native proteins include pKC30(Shimatake and Rosenberg, Nature 292:128, 1981), pKK177-3 (Amann andBrosius, Gene 40:183, 1985) and pET-3 (Studiar and Moffatt, J. Mol.Biol. 189:113, 1986), as well as the pCMV/R vector. The CMV/R promoteris described in, among other places, PCT Application No. PCT/US02/30251and PCT Publication No. WO03/028632.

The DNA sequence can also be transferred from its existing context toother cloning vehicles, such as other plasmids, bacteriophages, cosmids,animal viruses and yeast artificial chromosomes (YACs) (Burke et al.,Science 236:806-812, 1987). These vectors may then be introduced into avariety of hosts including somatic cells, and simple or complexorganisms, such as bacteria, fungi (Timberlake and Marshall, Science244:1313-1317, 1989), invertebrates, plants (Gasser and Fraley, Science244:1293, 1989), and animals (Pursel et al., Science 244:1281-1288,1989), which cell or organisms are rendered transgenic by theintroduction of the heterologous cDNA. Specific, non-limiting examplesof host cells include mammalian cells (such as CHO or HEK293 cells),insect cells (Hi5 or SF9 cells) or yeast cells.

For expression in mammalian cells, a cDNA sequence may be ligated toheterologous promoters, such as the simian virus (SV) 40 promoter in thepSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076,1981), or the cytomegalovirus promoter, and introduced into cells, suchas monkey COS-1 cells (Gluzman, Cell 23:175-182, 1981), to achievetransient or long-term expression. The stable integration of thechimeric gene construct may be maintained in mammalian cells bybiochemical selection, such as neomycin (Southern and Berg, J. Mol.Appl. Genet. 1:327-341, 1982) and mycophenolic acid (Mulligan and Berg,Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981).

DNA sequences can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR or other in vitroamplification.

A cDNA sequence (or portions derived from it) such as a cDNA encoding avariant HBsAg can be introduced into eukaryotic expression vectors byconventional techniques. These vectors are designed to permit thetranscription of the cDNA in eukaryotic cells by providing regulatorysequences that initiate and enhance the transcription of the cDNA andensure its proper splicing and polyadenylation. Vectors containing thepromoter and enhancer regions of the SV40 or long terminal repeat (LTR)of the Rous Sarcoma virus and polyadenylation and splicing signal fromSV40 are readily available (Mulligan et al., Proc. Natl. Acad. Sci. USA78:1078-2076, 1981; Gorman et al., Proc. Natl. Acad. Sci USA78:6777-6781, 1982). The level of expression of the cDNA can bemanipulated with this type of vector, either by using promoters thathave different activities (for example, the baculovirus pAC373 canexpress cDNAs at high levels in S. frugiperda cells (Summers and Smith,In Genetically Altered Viruses and the Environment, Fields et al. (Eds.)22:319-328, CSHL Press, Cold Spring Harbor, N.Y., 1985) or by usingvectors that contain promoters amenable to modulation, for example, theglucocorticoid-responsive promoter from the mouse mammary tumor virus(Lee et al., Nature 294:228, 1982). The expression of the cDNA can bemonitored in the recipient cells 24 to 72 hours after introduction(transient expression).

In addition, some vectors contain selectable markers such as the gpt(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981) orneo (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982) bacterialgenes. These selectable markers permit selection of transfected cellsthat exhibit stable, long-term expression of the vectors (and thereforethe cDNA). The vectors can be maintained in the cells as episomal,freely replicating entities by using regulatory elements of viruses suchas papilloma (Sarver et al., Mol. Cell Biol. 1:486, 1981) orEpstein-Barr (Sugden et al., Mol. Cell Biol. 5:410, 1985).Alternatively, one can also produce cell lines that have integrated thevector into genomic DNA. Both of these types of cell lines produce thegene product on a continuous basis. One can also produce cell lines thathave amplified the number of copies of the vector (and therefore of thecDNA as well) to create cell lines that can produce high levels of thegene product (Alt et al., J. Biol. Chem. 253:1357, 1978).

The transfer of DNA into eukaryotic, in particular human or othermammalian cells, is conventional. The vectors are introduced into therecipient cells as pure DNA (transfection) by, for example,precipitation with calcium phosphate (Graham and vander Eb, Virology52:466, 1973) or strontium phosphate (Brash et al., Mol. Cell Biol.7:2013, 1987), electroporation (Neumann et al., EMBO J 1:841, 1982),lipofection (Feigner et al., Proc. Natl. Acad. Sci USA 84:7413, 1987),DEAE dextran (McCuthan et al., J. Natl. Cancer Inst. 41:351, 1968),microinjection (Mueller et al., Cell 15:579, 1978), protoplast fusion(Schafner, Proc. Natl. Acad. Sci. USA 77:2163-2167, 1980), or pelletguns (Klein et al., Nature 327:70, 1987). Alternatively, the cDNA, orfragments thereof, can be introduced by infection with virus vectors.Systems are developed that use, for example, retroviruses (Bernstein etal., Gen. Engr'g 7:235, 1985), adenoviruses (Ahmad et al., J. Virol.57:267, 1986), or Herpes virus (Spaete et al., Cell 30:295, 1982).Polynucleotides that encode proteins, such as variant HBsAgs, can alsobe delivered to target cells in vitro via non-infectious systems, forinstance liposomes.

Using the above techniques, the expression vectors containing apolynucleotide encoding a variant HBsAgs as described herein or cDNA, orfragments or variants or mutants thereof, can be introduced into humancells, mammalian cells from other species or non-mammalian cells asdesired. The choice of cell is determined by the purpose of thetreatment. For example, monkey COS cells (Gluzman, Cell 23:175-182,1981) that produce high levels of the SV40 T antigen and permit thereplication of vectors containing the SV40 origin of replication may beused. Similarly, Chinese hamster ovary (CHO), mouse NIH 3T3 fibroblastsor human fibroblasts can be used.

The present disclosure, thus, encompasses recombinant vectors thatcomprise all or part of the polynucleotides encoding self-aggregatingvariant HBsAgs or cDNA sequences, for expression in a suitable host,either alone or as a labeled or otherwise detectable protein. The DNA isoperatively linked in the vector to an expression control sequence inthe recombinant DNA molecule so that the variant HBsAgs can beexpressed. The expression control sequence may be selected from thegroup consisting of sequences that control the expression of genes ofprokaryotic or eukaryotic cells and their viruses and combinationsthereof. The expression control sequence may be specifically selectedfrom the group consisting of the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the control region of fd coat protein, the early and latepromoters of SV40, promoters derived from polyoma, adenovirus,retrovirus, baculovirus and simian virus, the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, thepromoter of the yeast alpha-mating factors and combinations thereof.

Any host cell can be transfected with the vector of this disclosure.Exemplary host cells include, but are not limited to E. coli,Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus or otherbacilli; other bacteria; yeast; fungi; insect; mouse or other animal;plant hosts; or human tissue cells.

Multimeric forms of a variant HBsAg ring can be recovered (such as foradministration to a subject, or for other purposes) using any of avariety of methods known in the art for the purification of recombinantpolypeptides. The variant HBsAgs disclosed herein can producedefficiently by transfected cells and can be recovered in quantity usingany purification process known to those of skill in the art, such as anickel (NTA-agarose) affinity chromatography purification procedure.

A variety of common methods of protein purification may be used topurify the disclosed variant HBsAgs. Such methods include, for instance,protein chromatographic methods including ion exchange, gel filtration,HPLC, monoclonal antibody affinity chromatography and isolation ofinsoluble protein inclusion bodies after over production. In oneembodiment one or more purification affinity-tags, for instance asix-histidine sequence, is recombinantly fused to the protein, such asthe variant HBsAg, and used to facilitate polypeptide purification(optionally, in addition to another functionalizing portion of theprotein, such as a targeting domain or another tag, or a fluorescentprotein, peptide, or other marker).

Commercially produced protein expression/purification kits providetailored protocols for the purification of proteins made using eachsystem. See, for instance, the QIAEXPRESS™ expression system fromQIAGEN™ (Chatsworth, Calif.) and various expression systems provided byINVITROGEN™ (Carlsbad, Calif.). Where a commercial kit is employed toproduce a protein, such as a variant HBsAg, the manufacturer'spurification protocol is a preferred protocol for purification of thatprotein. For instance, proteins expressed with an amino-terminalhexa-histidine tag can be purified by binding to nickel-nitrilotriaceticacid (Ni-NTA) metal affinity chromatography matrix (TheQIAexpressionist, QIAGEN, 1997).

C. Therapeutic Methods and Pharmaceutical Compositions

Polynucleotides encoding the variant HBsAgs disclosed herein, andvariant HBsAgs, and the stable multimeric ring structures formed bypolypeptides expressed from such polynucleotides can be administered toa subject in order to generate an immune response to HIV-1. In oneexample, the immune response is a protective immune response. Thus, thepolynucleotides and polypeptides disclosed herein can be used in avaccine, such as a vaccine to prevent subsequent infection with HIV.

A therapeutically effective amount of variant HBsAg, a polymeric formthereof, a viral particle including these variant HBsAgs, or apolynucleotide encoding one or more of these polypeptides can beadministered to a subject to prevent, inhibit or to treat a condition,symptom or disease, such as acquired immunodeficiency syndrome (AIDS).In one example, polymeric ring structures formed by variant HBsAgssubunits are administered. In another example, one or morepolynucleotides encoding at least one variant HBsAgs is administered. Assuch, the variant HBsAgs and polynucleotides encoding variant HBsAgs canbe administered as vaccines to prophylactically or therapeuticallyinduce or enhance an immune response. For example, the pharmaceuticalcompositions described herein can be administered to stimulate aprotective immune response against HIV, such as a HIV-1.

A single administration can be utilized to prevent or treat an HIVinfection, or multiple sequential administrations can be performed. Inanother example, more than one of the variant HBsAgs, multimeric formsof more than one variant HBsAgs, or multiple polynucleotides encodingthe variant HBsAgs, are administered to a subject to induce an immuneresponse to HIV-1. These polypeptides or polynucleotides can beadministered simultaneously, or sequentially.

In exemplary applications, compositions are administered to a subjectinfected with HIV, or likely to be exposed to an infection, in an amountsufficient to raise an immune response to HIV. Administration induces asufficient immune response to reduce viral load, to prevent or lessen alater infection with the virus, or to reduce a sign or a symptom of HIVinfection. Amounts effective for this use will depend upon variousclinical parameters, including the general state of the subject'shealth, and the robustness of the subject's immune system, amongst otherfactors. A therapeutically effective amount of the compound is thatwhich provides either subjective relief of one or more symptom(s) of HIVinfection, an objectively identifiable improvement as noted by theclinician or other qualified observer, a decrease in viral load, anincrease in lymphocyte count, such as an increase in CD4 cells, orinhibition of development of symptoms associated with infection.

The variant HBsAgs, multimeric forms of the variant HBsAgs andpolynucleotides encoding them can be administered by any means known toone of skill in the art (see Banga, A., “Parenteral Controlled Deliveryof Therapeutic Peptides and Proteins,” in Therapeutic Peptides andProteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) such asby intramuscular, subcutaneous, or intravenous injection, but even oral,nasal, or anal administration is contemplated. Variant HBsAgs, polymericforms thereof, viral particles including the variant HBsAgs, orpolynucleotides encoding the variant HBsAgs can be administered in aformulation including a carrier or excipient. A wide variety of suitableexcipients are known in the art, including physiological phosphatebuffered saline (PBS), and the like. Optionally, the formulation caninclude additional components, such as aluminum hydroxylphophosulfate,alum, diphtheria CRM₁₉₇, or liposomes. To extend the time during whichthe peptide or protein is available to stimulate a response, the peptideor protein can be provided as an implant, an oily injection, or as aparticulate system. The particulate system can be a microparticle, amicrocapsule, a microsphere, a nanocapsule, or similar particle. Aparticulate carrier based on a synthetic polymer has been shown to actas an adjuvant to enhance the immune response, in addition to providinga controlled release. Aluminum salts may also be used as adjuvants toproduce an immune response.

In one embodiment, the variant HBsAgs or multimeric form thereof ismixed with an adjuvant containing two or more of a stabilizingdetergent, a micelle-forming agent, and an oil. Suitable stabilizingdetergents, micelle-forming agents, and oils are detailed in U.S. Pat.No. 5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202; andU.S. Pat. No. 5,695,770, all of which are incorporated by reference. Astabilizing detergent is any detergent that allows the components of theemulsion to remain as a stable emulsion. Such detergents includepolysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,ZWITTERGENT™3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, for example,Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977; and Hunter et al., J.Immunol 129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, andTETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. The chemicalstructures of such agents are well known in the art. In one embodiment,the agent is chosen to have a hydrophile-lipophile balance (HLB) ofbetween 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167,1984. The agent can be provided in an effective amount, for examplebetween 0.5 and 10%, or in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

An adjuvant can be included in the composition. In one example, theadjuvant is a water-in-oil emulsion in which antigen solution isemulsified in mineral oil (such as Freund's incomplete adjuvant ormontanide-ISA). In one embodiment, the adjuvant is a mixture ofstabilizing detergents, micelle-forming agent, and oil available underthe name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.). Otherexamples of suitable adjuvants are listed in the terms section of thisspecification.

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding one or more variant HBsAgs as disclosed herein. Atherapeutically effective amount of the immunogenic polynucleotide canbe administered to a subject in order to generate an immune response,such as a protective immune response.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding a variant HBsAg can beplaced under the control of a promoter to increase expression of themolecule. Suitable vectors are described, for example, in U.S. Pat. No.6,562,376.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. No.5,593,972 and U.S. Pat. No. 5,817,637 (which describe operativelylinking a nucleic acid sequence encoding an antigen to regulatorysequences enabling expression). U.S. Pat. No. 5,880,103 describesseveral methods of delivery of nucleic acids encoding immunogenicpeptides or other antigens to an organism. The methods include liposomaldelivery of the nucleic acids, and immune-stimulating constructs, orISCOMS™, negatively charged cage-like structures of 30-40 nm in sizeformed spontaneously on mixing cholesterol and QUIL A™ (saponin).Protective immunity has been generated in a variety of experimentalmodels of infection, including toxoplasmosis and Epstein-Barrvirus-induced tumors, using ISCOMS™ as the delivery vehicle for antigens(Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen aslow as 1 μg encapsulated in ISCOMS™ have been found to produce Class Imediated CTL responses (Takahashi et al., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, a variantHBsAg as disclosed herein can also be expressed by an attenuated viralhost or vector, or a bacterial vector. Recombinant adeno-associatedvirus (AAV), herpes virus, retrovirus, or other viral vectors can beused to express the peptide or protein, thereby eliciting a CTLresponse.

In one embodiment, a nucleic acid encoding the variant HBsAgs isintroduced directly into cells. For example, the nucleic acid may beloaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's HELIOS™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites, including tissues subject toor in proximity to a site of infection. Dosages for injection areusually around 0.5 μg/kg to about 50 mg/kg, and typically are about0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration, would include about 0.1 μg to 10 mg of avariant HBsAg per subject per day. Dosages from 0.1 pg to about 100 mgper subject per day can be used, particularly if the composition isadministered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceuticals Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa. (1995).

The compositions can be administered, either systemically or locally,for therapeutic treatments, such as to treat an HIV infection. Intherapeutic applications, a therapeutically effective amount of thecomposition is administered to a subject infected with HIV, such as, butnot limited to, a subject exhibiting signs or symptoms of AIDS. Singleor multiple administrations of the compositions can be administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of the HIV infection withoutproducing unacceptable toxicity to the subject.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa. (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342 (1994); Tice & Tabibi, Treatise onControlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. NewYork, N.Y., pp. 315-339 (1992)).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. No.5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat.No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; andU.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat.No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.5,534,496).

D. Immunodiagnostic Reagents and Kits

In addition to the therapeutic methods provided above, any of thevariant HBsAgs disclosed herein can be utilized to produce antigenspecific immunodiagnostic reagents, for example, for serosurveillance.Immunodiagnostic reagents can be designed from any of the antigenicpolypeptide described herein. For example, the presence of serumantibodies to HIV can be monitored using the isolated immunogensdisclosed herein, such as to detect an HIV infection. Generally, themethod includes contacting a sample from a subject, such as, but notlimited to a blood, serum, plama, urine or sputum sample from thesubject with one or more of the variant HBsAgs disclosed herein (or apolymeric form thereof) and detecting binding of antibodies in thesample to the variant HBsAgs. The binding can be detected by any meansknown to one of skill in the art, including the use of labeled secondaryantibodies that specifically bind the antibodies from the sample. Labelsinclude radiolabels, enzymatic labels, and fluorescent labels.

Any such immunodiagnostic reagents can be provided as components of akit. Optionally, such a kit includes additional components includingpackaging, instructions and various other reagents, such as buffers,substrates, antibodies or ligands, such as control antibodies orligands, and detection reagents.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Variant HBsAg Including Gp41 MPR Form Virus LikeParticles

This example illustrates variant HBsAg constructs that are capable offorming virus like particles.

Variant HBsAg were prepared by substituting the MPR and/or membranespanning domain of gp41 for various regions of the HBsAg (as illustratedin FIG. 2B), so that a gp41 MPR would be closely associated with thelipid layer of the particles (as illustrated in FIG. 1). For example, atthe amino end of HBsAg, the first construct (TM12) included the externalMPR domain of gp41 linked to the complete membrane spanning domain ofHBsAg (FIG. 3A). The second construct (TM14) linked a MPR to HBsAg at ajunction within the membrane spanning domain: it included six aminoacids from the TM domain of gp41 and up to 14 amino acids from themembrane spanning domain of HBsAg (FIG. 3A). The third construct (TM16)linked MPR plus the entire TM domain of gp41 to HBsAg without any of itsmembrane spanning domain (FIGS. 2A and 3A). All three constructs containantigenic determinants recognized by two monoclonal antibodies to gp41(see, for example, FIG. 10), although they may differ in their contactswith the lipid surface. In the exposed middle loop of variant HBsAgconstucts, the constructs expressed either the 2F5 epitope alone (TM32)or both the 2F5 and 4E10 epitopes (TM34). A third construct (TM32F)expressed four copies of the 2F5 epitope in tandem. In construct TM20(FIG. 9A), the entire third membrane spanning domain of HBsAg wasreplaced with the transmembrane domain of gp41, while including theepitopes for 2F5 and 4E10.

In one example, a variant HBsAg construct (TM16) was prepared bysubstituting the membrane spanning domain of gp41 for the entire firstmembrane spanning domain of HBsAg (see FIG. 2A). This construct waspredicted to anchor the MPR in the lipid membrane so that it would beoriented in a manner similar to its native orientation in gp41. Studieswere then performed to determine if MPR particles could be expressedwith different gp41 sequences substituted for the first membranespanning domain of HBsAg. FIG. 3A provides a schematic illustrating theinsertion of various gp41 membrane spanning domains into the firstmembrane spanning domain of HBsAg. FIG. 3B is a digital image of a gelillustrating MPR expression in HBsAg when gp41 is substituted for thebeginning of the first transmembrane domain of HBsAg (TM12) orsubstituted for the entire first transmembrane domain of HBsAg (TM16).However, substitution for approximately half of the first transmembranedomain of HBsAg (TM14) did not result in detectable MPR expression.Studies were then performed to determine the purity of TM16 (see FIG. 5)and if TM16 could form virus-like particles (see FIG. 6). TM16 waspurified by sedimentation in sucrose gradients, followed by hydrophobicinteraction chromatography, and the product was examined by electronmicroscopy (FIG. 6). TM16 particles are polymorphic in size, with arange 30+/−3 nm. This is similar to the size and heterogeneity of nativeHBsAg particles. Alternatively, TM16 was purified by banding in CsCldensity gradients, and this also demonstrated particles.

The HBsAg variant TM20 was generated in which the third transmembranedomain of HBsAg was substituted with a gp41 membrane spanning domain.The entire gene for the TM20 construct was synthesized and then insertedinto pFastbac cloning vector (commercially available from Invitrogen).Cells containing the complete baculovirus genome as a “bacmid” weretransfected and the TM20 coding sequence entering the bacmid via atransposon. Bacmid DNA was then transfected into Sf9 cells, resulting inthe production of baculovirus expressing the TM20 gene. Cell pelletswere screened for protein expression by Western blot analysis. The highexpressing baculovirus was grown to 200 ml stock and titered for PFU/ml.To express the protein, two to four large flasks of Hi 5 insect cells(200 ml each) were grown, at 1.5×10⁶/ml, and infect them at an moi of4:1. The cells were then incubated at 27° C. on a shaker, and harvestedat 28 to 30 hours. The cells were spun down, and the virus-likeparticles remained in the cells. The pellet from each flask was frozenin 10 ml phosphate buffered saline (PBS). The cells were lysed byfreezing and thawed followed by sonication for 1 min. The lysate (10 to20 ml) was layered onto a step gradient consisting of 10, 20 and 50%sucrose. Sedimentation was for 2 hrs and 20 min at 27,000 rpm in aBeckman SW28 rotor.

Fractions (0.5 ml each) were dripped from the bottom, and each fractionwas assayed for antigen content by ELISA. Active fractions were pooled,and the sample was stored frozen at −80° C. Further purification is doneby hydrophobic interaction chromatography. Briefly, 20 ml of pooledsucrose fractions were diluted to 30 ml with PBS, and ammonium sulfatewas added to make 0.7 M. Triton X100 was added to 0.025%, close to theCMC. The sample was passed twice through a 4 to 5 ml column ofMacro-Prep Methyl HIC support from Bio-Rad. Pass through fractions werecollected, followed by three wash fractions of one column volume each.Elution was performed in 2×PBS, and 8 fractions of 0.5 column volumewere collected. Each fraction is assayed by ELISA, and the peak elutionusually occurred in the second elution fraction. Antigen content wasdetermined by Western blot. Antigen quantity was then determined bycomparing antigen test sample to a known standard or by use of a digitalreader based on fluorescence.

TM20 particles were banded in CsCl and examined by electron microscopy.The particles were somewhat larger than TM16 particles, with a diameterof 45+/−5 nm (FIG. 6). Size exclusion chromatography and cesium chloridegradient studies (FIG. 7) demonstrated that TM20 particles assembledcorrectly (e.g., similar to native HBsAgs) despite replacing the thirdtransmembrane domain of HBsAg with a gp41 antigenic insert (such as theantigenic insert provided in FIG. 10).

FIG. 8 illustrates purification of an additional variant HBsAg (MPRS) bya methyl HIC column in which the variant HBsAg includes a gp41 insertfollowing the fourth domain of the HBsAg. The following additionalvariant HBsAgs were constructed and determined to be form virus-likeparticles: TM34, TM32F, TM16+20 and TM16+34 (see FIGS. 9A and 9B). TM32FHBsAg variant includes four MPRs in a row in the middle loop of HBsAg(FIG. 9B). TM16+20 HBsAg variant has a gp41 spanning membrane domain inthe first and third domain and two MPRs. TM16+34 has a MPR anchored to agp41 spanning membrane domain in the first domain of HBsAg and a MPR inthe middle loop of HBsAg (in between the second and third domain ofHBsAg), as illustrated in FIG. 9B. The constructs with two MPRs weredetermined to be more antigenic than any of tested single constructs.

These studies demonstrate that HBsAg particles allow varioussubstitutions within HBsAgs, such as within HBsAg membrane spanningdomains while still forming antigenic particles.

Expression of the three HBsAg hybrids with a MPR linked to the amino endwas tested by western blot (FIG. 3B), for two DNA constructs of eachtype. TM12 was expressed well, while TM14 was not expressed at all. Incontrast, TM16 was expressed as well as TM12, even though the entirefirst membrane spanning domain of HBsAg was replaced by thetransmembrane domain of gp41. A strong band on western blot generallyindicates particle assembly, since unassembled HBsAg is quicklydegraded. To evaluate this, each particle was sedimented in sucrosegradients and the peaks were detected with a monoclonal antibody to theMPR. As shown in FIG. 11 (left panel), TM12 and TM16 sedimented at largesize, comparable to native HBsAg particles. These results indicate thatHBsAg particles could assemble normally, even when the entire firstmembrane spanning domain was replaced by another transmembrane sequence.

The size and expression of HBsAg hybrids with a MPR linked to theexposed middle domain was tested by sedimentation in sucrose gradients(FIG. 11, right panel). These particles, TM32, TM34, and TM32F,sedimented at large size, similar to native HBsAg. This indicates thatthe MPR determinant could be expressed close to the major antigenicdeterminant of HBsAg, without disrupting particle formation. Extensionof TM34 to include the entire third membrane spanning domain producedTM20. This construct also formed particles, as shown by sedimentation atlarge size (FIG. 11, right side).

Next, the ability to form particles when two MPR substitutions in thesame construct was tested. Two of these, TM16+32F and TM16+34, combineda substitution at the first membrane spanning domain with one in theexposed middle loop (FIG. 9B). This left three membrane spanning domainsof HBsAg, and they all formed particles, as shown by sedimentation atlarge size in sucrose gradients. After purification by banding in CsCl,these particles were significantly larger by EM than native HBsAgparticles, with diameters ranging from 54+/−8 nm for TM16+32F to 55+/−7nm for TM16+34 (FIG. 12).

The construct most challenging for particle formation combined TM16 andTM20 in the same hybrid protein, called TM16+20. Despite the absence ofthe first and third membrane spanning domains of HBsAg, this constructsedimented at large size and formed particles, as shown by electronmicroscopy (FIG. 12). The two remaining HBsAg domains (domains 2 and 4)were sufficient for particle formation, and this was not blocked by twocopies of the transmembrane domain of gp41. After banding in CsCl,TM16+20 particles were studied by electron microscopy; they gave largeparticles, with a diameter of 52+/−8 nm (FIG. 12).

The HBsAg variant particles allowed the immunological properties of theMPR determinant to function when displayed on a lipid surface to beevaluated. By comparing antibody binding to homologous constructs, theeffect of MPR valency on antigenicity and immunogenicity weredetermined. For example, the initial constructs, such as TM12, TM16,TM20, and TM34, were monovalent in MPR, but the combined constructs,TM16+20 and TM16+34, were divalent in MPR, and TM16+32F was pentavalent.

Two monovalent MPR particles, TM16 and TM34, were combined to producedivalent TM16+34 particles. The binding of monoclonal 2F5 to thedivalent construct was compared to the monovalent parent constructs TM16and 34 or to a negative control of HBsAg (FIG. 13). Divalent insertionof MPR determinants into TM16+34 had a synergistic effect on antibodybinding: it was drastically greater than to the monovalent parentalforms, and it was greater than the sum of the monovalent antigens. Itwas also greater than binding to a recombinant gp140 control. Theseresults suggest cooperative binding of 2F5 to divalent MPR antigens,possibly by allowing both arms of the antibody to bind the same antigen.

Two additional MPR constructs, monovalent TM16 and MPR tetravalentTM32F, were combined to form the pentavalent construct, called TM16+32F.As before, antibody binding to the pentavalent MPR was greater thaneither of the parental forms or to the HBsAg negative control (FIG. 13).It was also greater than the sum of the parental forms. These resultssuggest cooperative binding of divalent antibodies to multivalent MPRparticles.

To determine the immunogenicity of the disclosed constructs, pairs ofrabbits were immunized with each of the multivalent particles (FIG. 14).The resulting antibodies were titered on ELISA plates coated with AT-2inactivated virions of the MN strain (FIG. 14, left panel). TM16+32F wasmore immunogenic than any other construct and 2/2 rabbits (B9 and B10)gave antibodies to MPR after the second dose. These antibodies werespecific for viral antigens, since there was no binding to amicrovessicle control. The greater immunogenicity of the TM16+32Fconstruct may be due to its greater valency, five versus two for theothers. AT-2 inactivated virions were employed in the ELISA assaybecause it contains native MPR antigen on a viral surface.

The rabbit sera were tested for cross reactivity on other HIV strains(aldrithiol-2 inactivated), or on a microvessicle control (FIG. 14,right panel). Strain 89.6 was the immunizing strain, but rabbit B9showed better cross reactive binding on all other strains tested. Itscross reactivity was comparable to that of monoclonal 2F5. In contrast,rabbit B10, with a lower antibody titer after two doses of antigen,bound only to the MN strain. Monoclonal 2F5 also bound MN better thanthe other strains, suggesting that MN virions may expose MPR better thanthe other strains. Pentavalent™ 16+32F particles can elicit antibodiesto a membrane proximal region determinant that is shared among a varietyof HIV isolates.

Therefore, these studies demonstrate that the transmembrane domain ofgp41 could be substituted for the entire first membrane spanning domainof HBsAg, while producing a hybrid protein that still formed particles.Particles could also be formed when the transmembrane domain of gp41 wasswapped for the third membrane spanning domain of HBsAg, and when it wassubstituted for both the first and third domains in the same construct.These particles demonstrated the plasticity and tenacity of particleformation by HBsAg. They also displayed the MPR determinant of HIV in away that closely resembles its natural form on the surface of virions.These determinants were displayed on a lipid surface, where they areanchored via their own transmembrane domain. They elicited antibodiescapable of binding HIV virions in a broadly crossreactive pattern. Assuch, these studies suggest that immunizing with a MPR determinant thatmimics its natural form on the surface of virions can elicit antibodiesthat bind this potent and cross reactive neutralizing determinant on thevirus.

It is believed that these particles express neutralizing determinants ofgp41 in a more exposed form, thereby providing support for use of theseparticles to produce new vaccine antigens with the potential to improvethe quantity and quality of the antibody response to HIV proteins.

Example 2 Treatment of HIV in a Human Subject

This example describes a particular method that can be used to treat HIVin a human subject by administration of one or more compositions thatincludes an effective amount of any of the disclosed isolatedimmunogens. Although particular methods, dosages, and modes ofadministrations are provided, one skilled in the art will appreciatethat variations can be made without substantially affecting thetreatment.

Based upon the teaching disclosed herein, HIV, such as HIV type 1, canbe treated by administering a therapeutically effective amount of acomposition that includes variant HBsAgs to reduce or eliminate HIVinfection, replication or a combination thereof. The method can includescreening subjects to determine if they have HIV, such as HIV-1.Subjects having HIV are selected. In one example, subjects havingincreased levels of HIV antibodies in their blood (as detected with anenzyme-linked immunosorbent assay, Western blot, immunofluorescenceassay, or nucleic acid testing, including viral RNA or proviral DNAamplification methods are selected. In one example, a clinical trialwould include half of the subjects following the established protocolfor treatment of HIV (such as a highly active antiretroviral therapy).The other half would follow the established protocol for treatment ofHIV (such as treatment with highly active antiretroviral compounds) incombination with administration of the compositions including variantHBsAgs (as described above). In another example, a clinical trial wouldinclude half of the subjects following the established protocol fortreatment of HIV (such as a highly active antiretroviral therapy). Theother half would receive a composition including variant HBsAgs (such asvirus-like particles including any of the disclosed variant HBsAgs, suchas variant HBsAg TM16, variant HBsAg TM20, variant HBsAg MPRS, variantDA31-34, variant DA31-32F, variant TM16+20, variant TM16+31/34 or anycombination thereof).

Screening Subjects

In particular examples, the subject is first screened to determine ifthey have HIV. Examples of methods that can be used to screen for HIVinclude a combination of measuring a subject's CD4+ T cell count and thelevel of HIV in serum blood levels.

In some examples, HIV testing consists of initial screening with anenzyme-linked immunosorbent assay (ELISA) to detect antibodies to HIV,such as to HIV-1. Specimens with a nonreactive result from the initialELISA are considered HIV-negative unless new exposure to an infectedpartner or partner of unknown HIV status has occurred. Specimens with areactive ELISA result are retested in duplicate. If the result of eitherduplicate test is reactive, the specimen is reported as repeatedlyreactive and undergoes confirmatory testing with a more specificsupplemental test (e.g., Western blot or an immunofluorescence assay(IFA)). Specimens that are repeatedly reactive by ELISA and positive byIFA or reactive by Western blot are considered HIV-positive andindicative of HIV infection. Specimens that are repeatedlyELISA-reactive and occasionally provide an indeterminate Western blotresult, which may be either an incomplete antibody response to HIV in aninfected subject, or nonspecific reactions in an uninfected subject. IFAcan be used to confirm infection in these ambiguous cases. In someinstances, a second specimen will be collected more than a month laterand retested for subjects with indeterminate Western blot results. Inadditional examples, nucleic acid testing (e.g., viral RNA or proviralDNA amplification method) can also help diagnosis in certain situations.

The detection of HIV in a subject's blood is indicative that the subjecthas HIV and is a candidate for receiving the therapeutic compositionsdisclosed herein. Moreover, detection of a CD4+ T cell count below 350per microliter, such as 200 cells per microliter, is also indicativethat the subject is likely to have HIV.

Pre-screening is not required prior to administration of the therapeuticcompositions disclosed herein.

Pre-Treatment of Subjects

In particular examples, the subject is treated prior to administrationof a therapeutic composition that includes one or more of the disclosedvariant HBsAgs. However, such pre-treatment is not always required, andcan be determined by a skilled clinician. For example, the subject canbe treated with an established protocol for treatment of HIV (such as ahighly active antiretroviral therapy).

Administration of Therapeutic Compositions

Following subject selection, a therapeutic effective dose of thecomposition including variant HBsAgs is administered to the subject(such as an adult human or a newborn infant either at risk forcontracting HIV or known to be infected with HIV). For example, atherapeutic effective dose of a composition including one or more of theHBsAg variants is administered to the subject to in an amount sufficientto raise an immune response to HIV. Administration induces a sufficientimmune response to reduce viral load, to prevent or lessen a laterinfection with the virus, or to reduce a sign or a symptom of HIVinfection. Additional agents, such as anti-viral agents, can also beadministered to the subject simultaneously or prior to or followingadministration of the disclosed compositions. Administration can beachieved by any method known in the art, such as oral administration,inhalation, intravenous, intramuscular, intraperitoneal, orsubcutaneous.

In some particular examples, the composition includes variant HBsAgswith one or more transmembrane domains of the HBsAg replaced with a gp41antigenic insert. The gp41 antigenic insert includes (a) an antigenicpolypeptide fragment of gp41, such as an antigenic polypeptide gp41fragment with the amino acid sequence of SEQ ID NO: 1, and (b) atransmembrane domain of gp41, such as a transmembrane spanning gp41region with the amino acid sequence set forth in SEQ ID NO: 25 (in whichwherein X₁, X₂, X₃ and X₄ are any amino acid X₅, X₆, and X₇ are anyhydrophobic amino acid). In one example, the antigenic polypeptidefragment of gp41 is between 28 and 150 amino acids in length and themembrane spanning region of gp41 is between 22 and 40 amino acids inlength.

In one particular example, the composition includes a variant HBsAg inwhich the first transmembrane spanning domain of the HBsAg is replacedby a gp41 antigenic insert. For example, the gp41 antigenic insertreplaces amino acid residues 1-35 of SEQ ID NO: 31. In another example,the gp41 antigenic insert replaces amino acid residues 1-32 of SEQ IDNO: 31. In yet another example, the gp41 antigenic insert replaces aminoacid residues 1-29 of SEQ ID NO: 31. In further examples, thecomposition includes a variant HBsAg in which the first transmembranespanning domain of the HBsAg is replaced by a gp41 antigenic insert andthe variant HBsAg has the amino acid sequence set forth as SEQ ID NO:29.

In another particular example, the composition includes includes avariant HBsAg in which the third transmembrane spanning domain of theHBsAg is replaced by a gp41 antigenic insert. For example, the gp41antigenic insert replaces amino acid residues 150-190 of SEQ ID NO: 31.In another example, the gp41 antigenic insert replaces amino acidresidues 153-187 of SEQ ID NO: 31. In yet another example, the gp41antigenic insert replaces amino acid residues 156-185 of SEQ ID NO: 31.In a further example, the composition includes a variant HBsAg in whichthe third transmembrane spanning domain of the HBsAg is replaced by agp41 antigenic insert and the variant HBsAg has the amino acid sequenceset forth as SEQ ID NO: 57.

In an even more particular example, the composition includes a variantHBsAg in which the first and the third transmembrane spanning domains ofthe HBsAg are replaced by a gp41 antigenic insert. For example, the gp41antigenic insert replaces amino acid residues 1-35 and 150-190 of SEQ IDNO: 31. In another example, the gp41 antigenic insert replaces aminoacid residues 1-32 and 153-187 of SEQ ID NO: 31. In yet another example,the gp41 antigenic insert replaces amino acid residues 1-29 and 156-185of SEQ ID NO: 31. In a particular example, the the composition includesa variant HBsAg in which the third transmembrane spanning domain of theHBsAg is replaced by a gp41 antigenic insert and the variant HBsAg hasthe amino acid sequence set forth as SEQ ID NO: 58.

In additional examples, the composition includes variant HBsAgs with agp41 transmembrane spanning domain inserted into the first domain andthird domain of the HBsAgs. In another example, the composition includesvariant HBsAgs with at least one MPR inserted into the HBsAg in betweenthe second domain and third domain. In additional examples, thecomposition includes a combination of the disclosed variant HBsAgs, suchas variant HBsAgs with a gp41 antigenic insert (including a gp41transmembrane domain and MPR) replacing the first and third domain ofthe HBsAg (variant HBsAg TM16+TM20) and variant HBsAgs with a gp41antigenic insert (a gp41 transmembrane domain and MPR) replacing thefirst domain of the HBsAg and a MPR in between the second and thirdtransmembrane domains (TM16+34). In other examples, the compositionincludes isolated nucleic acid molecules encoding the variant HBsAgs orviral-like particles including the varianat HBsAgs. In particularexamples, the composition includes variant HBsAgs with an amino acidsequence set forth by SEQ ID NOs: 56 and 62-64.

The amount of the composition administered to prevent, reduce, inhibit,and/or treat HIV or a condition associated with it depends on thesubject being treated, the severity of the disorder, and the manner ofadministration of the therapeutic composition. Ideally, atherapeutically effective amount of an agent is the amount sufficient toprevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV)in a subject without causing a substantial cytotoxic effect in thesubject. An effective amount can be readily determined by one skilled inthe art, for example using routine trials establishing dose responsecurves. In addition, particular exemplary dosages are provided above.The therapeutic compositions can be administered in a single dosedelivery, via continuous delivery over an extended time period, in arepeated administration protocol (for example, by a daily, weekly, ormonthly repeated administration protocol). In one example, therapeuticcompositions that include variant HBsAgs are administered intravenouslyto a human. As such, these compositions may be formulated with an inertdiluent or with an pharmaceutically acceptable carrier.

In one specific example, a composition including variant HBsAgs isadministered intravenously from 0.1 pg to about 100 mg per kg per day.In an example, the composition is administered continuously. In anotherexample, the composition is administered at 50 μg per kg given twice aweek for 2 to 3 weeks. Administration of the therapeutic compositionscan be taken long term (for example over a period of months or years).

Assessment

Following the administration of one or more therapies, subjects havingHIV (for example, HIV-1 or HIV-2) can be monitored for reductions in HIVlevels, increases in a subjects CD4+ T cell count, or reductions in oneor more clinical symptoms associated with HIV. In particular examples,subjects are analyzed one or more times, starting 7 days followingtreatment. Subjects can be monitored using any method known in the art.For example, biological samples from the subject, including blood, canbe obtained and alterations in HIV or CD4+ T cell levels evaluated.

Additional Treatments

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of agents that theypreviously received for the desired amount of time, including theduration of a subject's lifetime. A partial response is a reduction,such as at least a 10%, at least 20%, at least 30%, at least 40%, atleast 50%, or at least 70% in HIV infection, HIV replication orcombination thereof. A partial response may also be an increase in CD4+T cell count such as at least 350 T cells per microliter.

Example 3 Method of Monitoring Serum Antibodies to HIV

This example illustrates the methods of monitoring serum antibodies toHIV.

Based upon the teachings disclosed herein, the presence of serumantibodies to HIV can be monitored using the isolated immunogensdisclosed herein, such as to detect an HIV infection. Generally, themethod includes contacting a sample from a subject, such as, but notlimited to a blood, serum, plama, urine or sputum sample from thesubject with one or more of the variant HBsAgs disclosed herein (or apolymeric form thereof) and detecting binding of antibodies in thesample to the variant HBsAgs. The binding can be detected by any meansknown to one of skill in the art, including the use of labeled secondaryantibodies that specifically bind the antibodies from the sample. Labelsinclude radiolabels, enzymatic labels, and fluorescent labels.

The results can be used to distinguish a subpopulation of patients thatmake MPER antibodies in response to infection. This population may havea different clinical outcome, based on production of MPER-specificantibodies. They can also be used to detect a patient's immune responseto another vaccine antigen that elicits antibodies specific for the MPERdeterminant. The high responder patients detected in this way may be theones protected against HIV infection.

Example 4 Screening of Test Agents to Treat HIV

This example describes methods that can be used to identify agents totreat HIV.

According to the teachings herein, one or more agents for treating HIV,such as HIV-1, can be identified by contacting a cell, such as a cellexpressing a variant HBsAg, with one or more test agents underconditions sufficient for the one or more test agents to alter theactivity of the variant HBsAg. The method can include detectingalterations in HIV or CD4+ T cell levels. Various types of in vitroassays may be employed to identify agents to treat HIV including, butnot limited to, HIV-infection assays, LFA-activity assays, bindingassays, standard Western blot or immunoassay techniques and other wellknown assays to those of skill in the art. However, the disclosure isnot limited to particular methods of detection.

Regardless of the assay technique, agents that cause at least a 2-folddecrease, such as a 3-fold decrease, a 4-fold decrease, or 5-folddecrease in detectable HIV or at least a 2-fold increase, such as a3-fold increase, a 4-fold increase, or 5-fold increase in CD4+ T celllevels, are selected for further evaluation.

Potential therapeutic agents identified with these or other approaches,including the specific assays and screening systems described herein,are used as lead compounds to identify other agents having even greatermodulatory effects on HIV. Candidate agents also can be tested inadditional cell lines and animal models of HIV to determine theirtherapeutic value. The agents also can be tested for safety in animals,and then used for clinical trials in animals or humans. In one example,genetically engineered mouse models of HIV are employed to determinetherapeutic value of test agents. In another example, simianimmunodeficiency virus (SIV)-macaque or a chimeric simian-humanimmunodeficiency virus (SHIV)-macaque model are utilized. SHIV strainshave the viral envelope of HIV but the gag/pol genes of SIV.Pathogenesis is similar with respect to macrophage and T lymphocyte celltropism, histopathologic changes, CD4-cell depletion and clinical signsof AIDS in virulent strains. For example, a monkey is immunized withneutralizing antibodies and then challenged with a SHIV strain that isthe same as that used to vaccinate the animal. In another example, amonkey immunized with neutralizing antibodies and then challenged with aSHIV strain that is a different from the strain used to vaccinate theanimal. Such studies could be used to identify agents that would protectsubjects, such as humans, from HIV or reduce one or more symptomsassociated with HIV.

The results can be used to distinguish a subpopulation of patients thatmake MPER antibodies in response to infection. This population may havea different clinical outcome, based on production of MPER-specificantibodies. They can also be used to detect a patient's immune responseto another vaccine antigen that elicits antibodies specific for the MPERdeterminant. The high responder patients detected in this way may be theones protected against HIV infection.

Example 5 Binding of HIVIgG and Human Sera from HIV-1 Positive Patientsto Disclosed Variant HBsAg and Variant HBsAg Particles

Based upon the teaching herein, the utility of variant HBsAg particlesto identify sera that contain neutralizing antibodies against MPR can bedetermined by screening a set of weakly and broadly neutralizing humanHIV-1 positive sera and HIV-IgG for binding to variant HBsAg particles.Human sera from HIV-1 positive patients and antibody 2F5 can be seriallydiluted and analyzed for binding to variant HBsAg and variant HBsAgparticles in ELISA format. The results can be used to distinguish asubpopulation of patients that make MPER antibodies in response toinfection. This population may have a different clinical outcome, basedon production of MPER-specific antibodies. They can also be used todetect a patient's immune response to another vaccine antigen thatelicits antibodies specific for the MPER determinant. The high responderpatients detected in this way may be the ones protected against HIVinfection.

Example 6 Immunization of Rabbits with Variant HBsAg Particles

Based upon the teaching herein, rabbits are immunized with 5, 20, 50 and100 μg of the disclosed variant HBsAg particles in ALUM and CpG asadjuvant by intramuscular route. The rabbit sera is analyzed for bindingto HBsAg and 2F5 epitope-containing peptide by ELISA. In addition, thesera can be checked for their neutralizing ability in a viralneutralization assay using sensitive HIV-1 strains and chimeric HIV-2strains containing HIV-1 2F5 epitope. If the rabbits are immunized tovariant HBsAg particles, then a high titer of antibodies will be raisedto HBsAg and a 2F5 epitope.

TABLE 1 Various Oligonucleotide Primers Primer  Name Primer SequenceSAg- 5′ GGAGCTCGTCGA CAGCAA 3′ Forward (SEQ ID NO: 38) SAg- 5′GC TCT AGA CCC GA T GTA CAC CCA 3′ Reverse (SEQ ID NO: 39) MPR  5′GC TCT AGA AAC GAG CAG GAG CTG CTG 3′ Forward (SEQ ID NO: 40) MPR  5′CGC GGA TCC TCA CCC CTT GAT GTA CCA  Reverse CAG CCA CTT 3′(SEQ ID NO: 41) MPR-Foldon 5′ CGC GGA TCC TCA ATG GTG ATG GTG ATG  RevGTG GGG 3′(SEQ ID NO: 42) C-heptad- 5′GC TCT AGA GCC GTG GAG CGG TAC CTG 3′ MPR (SEQ ID NO: 43) ForwardMPR-Tm5 5′ CTCGGATCCTCAAATCATGATGAAAATCTTGAT 3′ Reverse (SEQ ID NO: 44)MPR-Tm10 5′ CTCGGATCCTCACACCAGGCCACCAACAAT 3′ Reverse (SEQ ID NO: 45)MPR-Tm15 5′ CTCGGATCCTCACACCAGCCTCAGGCCCAC 3′ Reverse (SEQ ID NO: 46)MPR-Tm23- 5′ CTCGGATCCTCAGGCGGGCGC 3′ C9 (SEQ ID NO: 47) Reverse AgeI 5′ CCCTGCAAGACCTGCACC Forward ACCACCGGTCAGGGCAACTCCAAGTTCCCC 3′(SEQ ID NO: 48) AgeI  5′ GGGGAACTTG GAGTTGCCCT GACCGGTGGT reverseGGTGCAGGTC TTGCAGGG 3′ (SEQ ID NO: 49) MPR AgeI 5′GGC ACC GGT AAC GAG CAG GAG CTG  Forward CTG 3′ (SEQ ID NO: 50) MPR AgeI5′ GGC ACC GGT CCC CTT GAT GTA CCA CAG  Reverse CCA CTT 3′(SEQ ID NO: 51) MPR SAG 5′ AGC GAA TTC AAC GAG CAG GAG CTG  ForwardCTG 3′ (SEQ ID NO: 52) MPR SAG 5′ CGC GGA TCC TCA CCC GA T GTA CAC Reverse CCA 3′ (SEQ ID NO: 53) SAG MPR RI 5′CAG GAA GCC GGA GGT GATGAA CCC CTT  forward GAT GTA CCA CAG CCA CTT 3′(SEQ ID NO: 54) SAG MPR   5′ AAG TGG CTG TGG TAC ATC AAG GGG TTC  RIATC ACC TCC GGC TTC CTG 3′ Reverse (SEQ ID NO: 55)

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only examples of the invention and shouldnot be taken as limiting the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas my invention all that comes within the scope and spirit of theseclaims.

I claim:
 1. An isolated immunogen comprising a variant hepatitis Bsurface antigen, wherein the variant hepatitis B surface antigencomprises a hepatitis B surface antigen with one or more transmembranedomains of the hepatitis B surface antigen replaced with a HumanImmunodeficiency Virus Type 1 (HIV-1) gp41 antigenic insert, wherein thegp41 antigenic insert comprises: a) an antigenic polypeptide fragment ofa gp41 comprising the amino acid sequence of SEQ ID NO: 1(NEX₁X₂LLX₃LDKWASLWNWFDITNWLWYIX₄) wherein the antigenic polypeptidefragment of gp41 is between 28 and 150 amino acids in length; and b) atransmembrane membrane region of a gp41 comprising the amino acidsequence set forth as SEQ ID NO: 25 (X₅FIMIVGGLX₆GLRIVFTX₇LSIV), whereinthe transmembrane domain of gp41 is between 22 and 40 amino acids inlength and wherein the transmembrane domain of gp41 is C-terminal to theantigenic polypeptide fragment of gp41, wherein X₁, X₂, X₃, and X₄ areany amino acid and X₅, X₆, and X₇ are any hydrophobic amino acid.
 2. Theisolated immunogen of claim 1, wherein the variant hepatitis B surfaceantigen comprises a hepatitis B surface antigen with one or moretransmembrane domains of the hepatitis B surface antigen replaced withthe gp41 antigenic insert and at least one additional membrane proximalregion inserted between a second and third domain in the variant HBsAg,wherein the gp41 antigenic insert comprises: a) an antigenic polypeptidefragment of gp41 comprising the amino acid sequence of SEQ ID NO: 24(NEKELLELDKWASLWNWFDITNWLWYIR); and b) a transmembrane membrane regionof gp41 comprising the amino acid sequence set forth as SEQ ID NO: 27(LFIMIVGGLIGLRIVFTALSIV) wherein the transmembrane domain of gp41 isC-terminal to the antigenic polypeptide fragment of gp41; and whereinthe membrane proximal region inserted between the second and thirdtransmembrane domain of the variant hepatitis B surface antigen isreplaced with one or more antigenic polypeptide fragments comprising theamino acid sequence of SEQ ID NO: 60 (NEKELLELDKWASLW) or SEQ ID NO: 61(NEKELLELDKWASLWNWFDITNWL).
 3. The isolated immunogen of claim 1,wherein the antigenic polypeptide comprises the amino acid set forth asone of: a) SEQ ID NO: 2 (NEQELLALDKWASLWNWFDITNWLWYIK); b) SEQ ID NO: 3(NEQDLLALDKWASLWNWFDITNWLWYIK); c) SEQ ID NO: 4(NEQDLLALDKWANLWNWFDISNWLWYIK); d) SEQ ID NO: 5(NEQDLLALDKWANLWNWFNITNWLWYIR); e) SEQ ID NO: 6(NEQELLELDKWASLWNWFDITNWLWYIK); f) SEQ ID NO: 7(NEKDLLALDSWKNLWNWFDITNWLWYIK); g)  SEQ ID NO: 8(NEQDLLALDSWENLWNWFDITNWLWYIK); h) SEQ ID NO: 9(NEQELLELDKWASLWNWFSITQWLWYIK); i) SEQ ID NO: 10(NEQELLALDKWASLWNWFDISNWLWYIK); j)  SEQ ID NO: 11(NEQDLLALDKWDNLWSWFTITNWLWYIK); k) SEQ ID NO: 12(NEQDLLALDKWASLWNWFDITKWLWYIK); l) SEQ ID NO: 13(NEQDLLALDKWASLWNWFSITNWLWYIK); m) SEQ ID NO: 14(NEKDLLELDKWASLWNWFDITNWLWYIK); n) SEQ ID NO: 15(NEQEILALDKWASLWNWFDISKWLWYIK); o) SEQ ID NO: 16(NEQDLLALDKWANLWNWFNISNWLWYIK); p) SEQ ID NO: 17(NEQDLLALDKWASLWSWFDISNWLWYIK); q) SEQ ID NO: 18(NEKDLLALDSWKNLWSWFDITNWLWYIK); r) SEQ ID NO: 19(NEQELLQLDKWASLWNWFSITNWLWYIK); s) SEQ ID NO: 20(NEQDLLALDKWASLWNWFDISNWLWYIK); t) SEQ ID NO: 21(NEQELLALDKWASLWNWFDISNWLWYIR); u) SEQ ID NO: 22(NEQELLELDKWASLWNWFNITNWLWYIK); v) SEQ ID NO: 23(NEKELLELDKWASLWNWFDITNWLWYI): or w) SEQ ID NO: 24(NEKELLELDKWASLWNWFDITNWLWYIR).


4. The isolated immunogen of claim 1, wherein the transmembrane domainof gp41 comprises the amino acid set forth as one of: a)  SEQ ID NO: 26(IFIMIVGGLIGLRIVFTVLSIV) b)  SEQ ID NO: 27 (LFIMIVGGLIGLRIVFTALSIV);  orc)  SEQ ID NO: 28  (IFIMIVGGLVGLRIVFTALSIV).


5. The isolated immunogen of claim 1, wherein the hepatitis B surfaceantigen comprises a leader sequence as set forth by SEQ ID NO:
 30. 6.The isolated immunogen of claim 1, wherein a first transmembrane domainof the hepatitis B surface antigen is replaced by the gp41 transmembranedomain.
 7. The isolated immunogen of claim 6, wherein the gp41 antigenicinsert replaces amino acid residues 1-29 of SEQ ID NO: 31, amino acids1-32 of SEQ ID NO: 31, or amino acids 1-35 of SEQ ID NO:
 31. 8. Theisolated immunogen of claim 6, wherein the isolated immunogen comprisesthe amino acid sequence set forth as SEQ ID NO:
 29. 9. The isolatedimmunogen of claim 1, wherein a third transmembrane domain of thehepatitis B surface antigen is replaced by the gp41 transmembranedomain.
 10. The isolated immunogen of claim 9, wherein the gp41transmembrane domain replaces amino acid residues 150-190 of SEQ ID NO:31, amino acid residues 153-187 of SEQ ID NO: 31 or amino acid residues156-185 of SEQ ID NO:
 31. 11. The isolated immunogen of claim 9, whereinthe isolated immunogen comprises the amino acid sequence set forth asSEQ ID NO:
 57. 12. The isolated immunogen of claim 1, wherein a firsttransmembrane domain of the hepatitis B surface antigen is replaced bythe gp41 transmembrane domain and a third transmembrane domain of thehepatitis B surface antigen is replaced by the gp41 transmembranedomain.
 13. The isolated immunogen of claim 12, wherein the gp41transmembrane domain replaces amino acid residues 1-35 of SEQ ID NO: 31and 150-190 of SEQ ID NO: 31; or replaces amino acid residues 1-32 ofSEQ ID NO: 31 and 153-187 of SEQ ID NO:
 31. 14. The isolated immunogenof claim 12, wherein the isolated immunogen comprises the amino acidsequence set forth as SEQ ID NO:
 58. 15. The isolated immunogen of claim1, wherein a gp41 antigenic insert is inserted following a fourthmembrane domain of the hepatitis B surface antigen.
 16. The isolatedimmunogen of claim 1, wherein the variant HBsAg further comprises atleast one additional MPR.
 17. The isolated immunogen of claim 1, furthercomprising an HIV-specific T-helper cell epitope.
 18. The isolatedimmunogen of claim 1, further comprising the amino acid sequence setforth as SEQ ID NO: 34 (CAAX).
 19. A viral-like particle comprising theisolated immunogen of claim
 1. 20. The viral-like particle of claim 19,further comprising at least one TLR ligand.
 21. A composition comprisingan effective amount of the isolated immunogen of claim
 1. 22. Thecomposition of claim 21, further comprising an adjuvant.
 23. A methodfor inhibiting an HIV infection or for inducing an immune response toHIV in a subject, comprising administering an effective amount of thecomposition of claim 22 to the subject, thereby inhibiting HIV infectionor inducing the immune response, respectively.
 24. The method of claim23, wherein the at least one additional MPR is inserted between a seconddomain and a third domain of the variant HBsAg.
 25. The method of claim24, wherein the HIV-specific T-helper cell epitope comprises the aminoacid sequence set forth as SEQ ID NO: 33.